Incubation with either of the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and sn-1,2 dioctanoylglycerol (DiC8) decreased the uptake of dopamine into striatal synaptosomes, whereas the inactive phorbol ester 4 alpha-PMA had no effect. Washout of PMA and DiC8 failed to reverse the decrease in uptake. Kinetic analysis showed a decrease in the apparent V(max) for the transporter without changes in the K(m). Neither PMA nor DiC8 affected mazindol binding to the dopamine transporter. Preincubation with the protein kinase inhibitor staurosporine prevented the DiC8-induced decrease of dopamine uptake. Furthermore, the protein phosphatase inhibitor okadaic acid decreased dopamine uptake by itself and enhanced the DiC8-induced reduction of uptake. These findings support a role for protein kinase C in modulating dopamine transporter activity.