Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.