Mammalian D₁ and D₂ dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D₁-selective antagonist [³H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D₁ receptors, while saturable, dose-dependent, high affinity binding of the D₂-selective antagonist [³H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D₂ receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D₁ and D₂ receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D₁ receptor bound the (+)-enantiomer of the D₁-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D₂ receptor bound the (-)-enantiomer of the D₂-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPγS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D₁ and D₂ receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D₁ and D₂ dopamine receptors free of promiscuous G protein contaminants.