Subanesthetic ketamine is increasingly used for treating psychiatric conditions. While classified as an NMDAR antagonist, its mechanisms are incomplete. Recent evidence suggests its efficacy may involve opioid signaling. Using functional ultrasound imaging in rats, the study found that opioid receptor blockade suppressed ketamine-evoked neurophysiological changes in limbic regions, but not with a more selective NMDAR antagonist. This opioid-dependent response was sex-dependent, not evident in females and reversed by gonad removal in males. Similar effects were observed in postsynaptic density, behavioral sensitization, and opioid receptor density changes. These results indicate ketamine's affective responses may involve opioid signaling, influenced by sex. Future clinical trials should consider sex. Subanesthetic ketamine rapidly reduces depressive symptoms, leading to FDA approval for treatment-resistant depression. However, its mechanism remains unclear. Ketamine is commonly attributed to NMDAR antagonism, but other pathways may also be involved. Clinical trials show naltrexone reduces ketamine's antidepressant effect, but findings are conflicting. Preclinical data show mixed results, with some studies showing opioid receptor blockade suppresses ketamine responses, while others report no effect. The opioid system's role in ketamine's antidepressant effect raises concerns about abuse liability, as opioid signaling is linked to reward processing and drug reinforcement. This is especially relevant given the opioid crisis. Limited assessment of biological variables, including sex, may explain clinical and preclinical findings. The study used functional ultrasound imaging to assess ketamine's neural activity changes in awake-restrained rats. Ketamine evoked CBV signal increases in cortical and subcortical regions. Dose-response relationships were confirmed. PharmacofUSI closely tracked ketamine-evoked gamma-band power in the prefrontal cortex, confirming neural activity changes. Opioid receptor blockade modulated ketamine responses in males but not females. Naltrexone pretreatment reduced ketamine-induced activity in male regions like Cg1, M1/2, CPu, and NAc, while increasing activity in RSG, LHb, and LPLR. These effects were absent in females. Opioid receptor blockade also suppressed ketamine-evoked postsynaptic density changes and behavioral sensitization in males but not females. Naltrexone pretreatment blocked ketamine's effect in males but not females, suggesting male sex hormones gate the opioid-mediated response. In a different cohort, orchiectomy reversed the effect, indicating endocrine factors. Naltrexone did not affect MK-801-evoked CBV changes, suggesting effects are specific to ketamine. Opioid receptor blockade suppressed ketamine-induced postsynaptic density protein expression in males but not females. Ketamine-induced locomotor sensitization was opioid-mediated and sex-dependent, with naltrexone blocking the effect in males but not females. Chronic naltrexone increased mu opioid receptor density in females, potentially explaining the lack of effect in females. TheSubanesthetic ketamine is increasingly used for treating psychiatric conditions. While classified as an NMDAR antagonist, its mechanisms are incomplete. Recent evidence suggests its efficacy may involve opioid signaling. Using functional ultrasound imaging in rats, the study found that opioid receptor blockade suppressed ketamine-evoked neurophysiological changes in limbic regions, but not with a more selective NMDAR antagonist. This opioid-dependent response was sex-dependent, not evident in females and reversed by gonad removal in males. Similar effects were observed in postsynaptic density, behavioral sensitization, and opioid receptor density changes. These results indicate ketamine's affective responses may involve opioid signaling, influenced by sex. Future clinical trials should consider sex. Subanesthetic ketamine rapidly reduces depressive symptoms, leading to FDA approval for treatment-resistant depression. However, its mechanism remains unclear. Ketamine is commonly attributed to NMDAR antagonism, but other pathways may also be involved. Clinical trials show naltrexone reduces ketamine's antidepressant effect, but findings are conflicting. Preclinical data show mixed results, with some studies showing opioid receptor blockade suppresses ketamine responses, while others report no effect. The opioid system's role in ketamine's antidepressant effect raises concerns about abuse liability, as opioid signaling is linked to reward processing and drug reinforcement. This is especially relevant given the opioid crisis. Limited assessment of biological variables, including sex, may explain clinical and preclinical findings. The study used functional ultrasound imaging to assess ketamine's neural activity changes in awake-restrained rats. Ketamine evoked CBV signal increases in cortical and subcortical regions. Dose-response relationships were confirmed. PharmacofUSI closely tracked ketamine-evoked gamma-band power in the prefrontal cortex, confirming neural activity changes. Opioid receptor blockade modulated ketamine responses in males but not females. Naltrexone pretreatment reduced ketamine-induced activity in male regions like Cg1, M1/2, CPu, and NAc, while increasing activity in RSG, LHb, and LPLR. These effects were absent in females. Opioid receptor blockade also suppressed ketamine-evoked postsynaptic density changes and behavioral sensitization in males but not females. Naltrexone pretreatment blocked ketamine's effect in males but not females, suggesting male sex hormones gate the opioid-mediated response. In a different cohort, orchiectomy reversed the effect, indicating endocrine factors. Naltrexone did not affect MK-801-evoked CBV changes, suggesting effects are specific to ketamine. Opioid receptor blockade suppressed ketamine-induced postsynaptic density protein expression in males but not females. Ketamine-induced locomotor sensitization was opioid-mediated and sex-dependent, with naltrexone blocking the effect in males but not females. Chronic naltrexone increased mu opioid receptor density in females, potentially explaining the lack of effect in females. The