The presence and function of CB2 receptors in central nervous system (CNS) neurons have been controversial. This study reports the expression of CB2 receptor mRNA and protein localization on brainstem neurons. These functional CB2 receptors in the brainstem were activated by a CB2 receptor agonist, 2-arachidonoylglycerol, and by elevated endogenous levels of endocannabinoids, which also act at CB1 receptors. CB2 receptors represent an alternative site of action of endocannabinoids that opens the possibility of nonpsychotropic therapeutic interventions using enhanced endocannabinoid levels in localized brain areas. Endocannabinoids, anandamide, and 2-arachidonoylglycerol (2-AG) are lipid mediators that act at CB1 and CB2 receptors. Their actions are terminated through cellular uptake facilitated by a putative endocannabinoid transporter, followed by intracellular enzymatic hydrolysis. Two degradative enzymes for endocannabinoid metabolism are known; fatty acid amide hydrolase (FAAH) preferentially degrades anandamide, and monoacylglycerol lipase preferentially degrades 2-AG. The CB1 receptor is highly expressed in the CNS, where cannabinoids act at presynaptic CB1 receptors to elicit changes in the synaptic efficacy of neuronal circuits. The CB2 receptor has been found outside the CNS and is particularly associated with immune tissues, such as the spleen and thymus, as well as in various circulating immune cell populations. In the CNS, CB2 receptor mRNA has been reported in cerebellar granule cells, and CB2 receptors have been described on perivascular microglial cells and in cultured cerebrovascular endothelium. CB2 receptor expression is enhanced on glia in neuritic plaques and on immune cells in simian immunodeficiency virus encephalitis. The study found CB2 receptor mRNA expression in the brain (cerebellum, cortex, and brainstem) and spleen of the rat using reverse transcription polymerase chain reaction (RT-PCR). Sequencing of the 472-base pair PCR product showed that the products amplified from the spleen, cortex, and brainstem were identical to the rat CB2 receptor sequence. Using quantitative real-time RT-PCR, the brainstem contained 1.5 ± 0.9% of the CB2 receptor mRNA found in the spleen. Amplification of CB2 receptor from the brainstem was not due to genomic contamination of our sample, because amplification of RNA that was not reverse-transcribed did not lead to the generation of a product. Furthermore, our real-time PCR primers spanned intron-exon borders, which ensured that the only product that could be amplified was the spliced mRNA. The study investigated whether CB2 receptor protein could be detected by Western blotting and/or immunohistochemistry. Western blottingThe presence and function of CB2 receptors in central nervous system (CNS) neurons have been controversial. This study reports the expression of CB2 receptor mRNA and protein localization on brainstem neurons. These functional CB2 receptors in the brainstem were activated by a CB2 receptor agonist, 2-arachidonoylglycerol, and by elevated endogenous levels of endocannabinoids, which also act at CB1 receptors. CB2 receptors represent an alternative site of action of endocannabinoids that opens the possibility of nonpsychotropic therapeutic interventions using enhanced endocannabinoid levels in localized brain areas. Endocannabinoids, anandamide, and 2-arachidonoylglycerol (2-AG) are lipid mediators that act at CB1 and CB2 receptors. Their actions are terminated through cellular uptake facilitated by a putative endocannabinoid transporter, followed by intracellular enzymatic hydrolysis. Two degradative enzymes for endocannabinoid metabolism are known; fatty acid amide hydrolase (FAAH) preferentially degrades anandamide, and monoacylglycerol lipase preferentially degrades 2-AG. The CB1 receptor is highly expressed in the CNS, where cannabinoids act at presynaptic CB1 receptors to elicit changes in the synaptic efficacy of neuronal circuits. The CB2 receptor has been found outside the CNS and is particularly associated with immune tissues, such as the spleen and thymus, as well as in various circulating immune cell populations. In the CNS, CB2 receptor mRNA has been reported in cerebellar granule cells, and CB2 receptors have been described on perivascular microglial cells and in cultured cerebrovascular endothelium. CB2 receptor expression is enhanced on glia in neuritic plaques and on immune cells in simian immunodeficiency virus encephalitis. The study found CB2 receptor mRNA expression in the brain (cerebellum, cortex, and brainstem) and spleen of the rat using reverse transcription polymerase chain reaction (RT-PCR). Sequencing of the 472-base pair PCR product showed that the products amplified from the spleen, cortex, and brainstem were identical to the rat CB2 receptor sequence. Using quantitative real-time RT-PCR, the brainstem contained 1.5 ± 0.9% of the CB2 receptor mRNA found in the spleen. Amplification of CB2 receptor from the brainstem was not due to genomic contamination of our sample, because amplification of RNA that was not reverse-transcribed did not lead to the generation of a product. Furthermore, our real-time PCR primers spanned intron-exon borders, which ensured that the only product that could be amplified was the spliced mRNA. The study investigated whether CB2 receptor protein could be detected by Western blotting and/or immunohistochemistry. Western blotting