Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca2+-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKCδ, had no effect. A highly specific PKCβ inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorboI-13-acetate. None of the PKC inhibitors significantly altered [3H]dopamine uptake. PKCβI and PKCβII, but not PKCα or PKCγ, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKCβI and PKCβII but not PKCα or PKCγ were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKCβII as compared with hDAT-HEK 203 cells alone, or hDAT-HEK 293 cells transfected with PKCα or PKCβI. These results suggest that classical PKCβ II is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.