This study investigates the modulation of anxiety through the blockade of anandamide hydrolysis. Anandamide, an endogenous cannabinoid, is degraded by the enzyme fatty acid amide hydrolase (FAAH). The researchers developed potent, selective, and systemically active inhibitors of FAAH, which prevent the degradation of anandamide, thereby increasing its brain levels. These inhibitors, URB532 and URB597, exhibited anxiolytic-like effects in rats, as evidenced by increased time spent in open areas of the elevated zero-maze test and reduced isolation-induced vocalizations. These effects were accompanied by elevated brain levels of anandamide and were prevented by CB1 receptor blockade, suggesting that anandamide plays a role in emotional state modulation. The study also found that FAAH inhibitors, such as URB597, significantly increased brain anandamide levels without affecting those of the second endogenous cannabinoid, 2-AG. These inhibitors showed high selectivity for FAAH compared to other targets, including cannabinoid receptors and MGL. Despite their ability to inhibit FAAH and increase anandamide levels, the inhibitors did not produce the typical cannabinoid-related effects such as catalepsy, hypothermia, or hyperphagia, indicating that their anxiolytic effects are mediated through anandamide's tonic actions on CB1 receptors. The study highlights the potential of FAAH inhibition as a novel approach to anti-anxiety therapy, offering a way to enhance the effects of endogenous cannabinoids without the side effects associated with direct cannabinoid agonists. The findings suggest that anandamide contributes to the modulation of emotional states and that FAAH inhibitors could be a promising therapeutic strategy for anxiety disorders.This study investigates the modulation of anxiety through the blockade of anandamide hydrolysis. Anandamide, an endogenous cannabinoid, is degraded by the enzyme fatty acid amide hydrolase (FAAH). The researchers developed potent, selective, and systemically active inhibitors of FAAH, which prevent the degradation of anandamide, thereby increasing its brain levels. These inhibitors, URB532 and URB597, exhibited anxiolytic-like effects in rats, as evidenced by increased time spent in open areas of the elevated zero-maze test and reduced isolation-induced vocalizations. These effects were accompanied by elevated brain levels of anandamide and were prevented by CB1 receptor blockade, suggesting that anandamide plays a role in emotional state modulation. The study also found that FAAH inhibitors, such as URB597, significantly increased brain anandamide levels without affecting those of the second endogenous cannabinoid, 2-AG. These inhibitors showed high selectivity for FAAH compared to other targets, including cannabinoid receptors and MGL. Despite their ability to inhibit FAAH and increase anandamide levels, the inhibitors did not produce the typical cannabinoid-related effects such as catalepsy, hypothermia, or hyperphagia, indicating that their anxiolytic effects are mediated through anandamide's tonic actions on CB1 receptors. The study highlights the potential of FAAH inhibition as a novel approach to anti-anxiety therapy, offering a way to enhance the effects of endogenous cannabinoids without the side effects associated with direct cannabinoid agonists. The findings suggest that anandamide contributes to the modulation of emotional states and that FAAH inhibitors could be a promising therapeutic strategy for anxiety disorders.