Abstract Serotonin 2C receptors (5-HT2C R) appear to exert tonic inhibitory influence over dopamine (DA) neurotransmission in the ventral tegmental area (VTA), the origin of the mesolimbic DA system, thought to be important in psychiatric disorders including addiction and schizophrenia. Current literature suggests that the inhibitory influence of 5-HT2C R on DA neurotransmission occurs via indirect activation of GABA inhibitory neurons, rather than via a direct action of 5-HT2C R on DA neurons. The present experiments were performed to establish the distribution of 5-HT2C R protein on DA and GABA neurons in the VTA of male rats via double-label immunofluorescence techniques. The 5-HT2C R protein was found to be co-localized with the GABA synthetic enzyme glutamic acid decarboxylase (GAD), confirming the presence of the 5-HT2C R on GABA neurons within the VTA. The 5-HT2C R immunoreactivity was also present in cells that contained immunoreactivity for tyrosine hydroxylase (TH), the DA synthetic enzyme, validating the localization of 5-HT2C R to DA neurons in the VTA. While the degree of 5-HT2C R+GAD co-localization was similar across the rostro-caudal levels of VTA subnuclei, 5-HT2C R+TH co-localization was highest in the middle relative to rostral and caudal levels of the VTA, particularly in the paranigral, parabrachial, and interfascicular subnuclei. The present results suggest that the inhibitory influence of the 5-HT2C R over DA neurotransmission in the VTA is a multifaceted and complex interplay of 5-HT2C R control of the output of both GABA and DA neurons within this region.