Exposure of D1 dopamine receptors to agonists results in rapid desensitization of the receptor-stimulated accumulation of cAMP. It is believed that agonist-induced phosphorylation of the receptor plays a critical role in the processes that underlie this phenomenon. To investigate the role of agonist-induced receptor phosphorylation, a FLAG epitope was added to the amino terminus of the rat D1 dopamine receptor and this construct was stably expressed in C6 glioma cells. It was found that the D1 receptor was stoichiometrically phosphorylated under basal conditions and that its phosphorylation state was increased by 2- to 3-fold upon exposure of the cells to dopamine for 10 min. The dopamine-induced receptor phosphorylation could be blocked by D1-selective antagonists but was unaffected by inhibitors of either protein kinase A or protein kinase C. The incorporation of phosphate into the receptor was rapid but transient, despite the continued presence of dopamine. A comparison of the rates of receptor phosphorylation approximately ion (t(1/2) < 1 min) and dopamine-induced desensitization (t(1/2) approximately 7 min) revealed that receptor phosphorylation was not the rate limiting step for receptor desensitization. Upon removal of dopamine, the receptor was rapidly dephosphorylated (t(1/2) approximately 10 min) and this was not blocked by agents (i.e., concanavalin A or hypertonic sucrose) that inhibit D1 receptor internalization. Using specific inhibitors, the phosphatase involved in D1 receptor dephosphorylation was shown not to correlate with the recently identified "G protein-coupled receptor phosphatase" (Proc Natl Acad Sci USA 92:8343-8347, 1995). These results suggest that the phosphorylated D(1) receptor is processed through a novel recovery pathway and that internalization is not required for receptor dephosphorylation.