Dopamine transporters (DATs) undergo increased phosphorylation upon treatment of striatal tissue or cultured cells with protein kinase C activators and protein phosphatase inhibitors. Phosphorylation conditions also lead to reductions in dopamine transport activity, which may function to regulate synaptic dopamine levels and control the extent and duration of dopaminergic signaling. Treatment of rat striatal tissue with okadaic acid (OA), a broad-spectrum protein phosphatase inhibitor, produces apparent maximal increases in DAT phosphorylation, suggesting that dephosphorylation is a crucial regulator of the DAT phosphorylation state. We used a combination of endogenous and in vitro approaches to identify the phosphatase(s) responsible for this activity. In homogenates prepared from (32)PO(4)-labeled rat striatal slices, OA inhibited DAT dephosphorylation with an IC(50) of 40 nM, a dose most compatible with inhibition of protein phosphatase 1 (PP1). Dephosphorylation of DAT in striatal homogenates was also inhibited by PP1 inhibitor 2, while little effect was produced by protein phosphatase 2A inhibitor 1. In vitro dephosphorylation assays showed substantial removal of (32)PO(4) from DATs by PP1 but not by protein phosphatase 2A, protein phosphatase 2B, or protein tyrosine phosphatase, and this effect was blocked by OA, verifying that the (32)PO(4) loss from DAT was due to dephosphorylation. These results demonstrate that DAT is a direct substrate for PP1 in vitro and suggest that PP1 is a major DAT phosphatase in rat striatum.