The dopamine transporter (DAT) is a critical regulator of dopaminergic neurotransmission. Research in both rat striatum and heterologous cells suggests that protein kinase C beta (PKCbeta) is important for proper trafficking of DAT. However, a critical gap that is missing from the literature is the localization of PKCbeta to mesencephalic dopaminergic neurons. In this study we examined the co-localization of DAT, which serves to identify dopaminergic neurons, and PKCbeta in mesencephalic dopaminergic cells. Using immunofluorescence and confocal microscopy, we demonstrated co-localization of DAT and PKCbeta in primary cultures of mesencephalic neurons and in dopamine neurons in rat substantia nigra and ventral tegmental area. PKCbetawas not specific for dopamine neurons in the two brain regions. This is the first demonstration of co-localization of PKCbeta and DAT in mesencephalic neurons. The co-localization of PKCbeta with DAT in mesencephalic neurons corroborates our previous studies demonstrating a role for PKCbeta in DAT function.