A defect in the coupling of the D(1) receptor (D(1)R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D(1)R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D(1)R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3',5' monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D(1)Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0+/-1.5 density units, DU) than in normotensive rats (7.4+/-2.9 DU). The increased basal serine phosphorylation of D(1)Rs in SHRs was accompanied by decreased expression of D(1)R at the cell surface, and decreased ability of a D(1)-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3',5' monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17+/-4%) and alter D(1)R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D(1)R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26+/-6%) in SHRs. Uncoupling of the D(1)R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D(1)R serine phosphorylation.