In the membranes of Spodoptera frugiperda (Sf-9) insect cells heterologously expressing the human D3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [35S]GTPgammaS binding. Quinpirole, for instance, dose-dependently enhanced [35S]GTPgammaS binding with a half-maximal concentration of 2.3 +/- 0.2 nM. Its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, YM-09151-2. A number of known agonists enhanced [35S]GTPgammaS binding to variable degrees, probably reflecting their differential efficacy to activate target G-proteins via the receptor. This agonist-induced [35S]GTPgammaS binding was abolished by N-ethylmaleimide, a selective blocking agent for Gi/Go proteins, with no appreciable effect on ligand binding. We propose coupling of the cloned D3 receptor to endogenous G-proteins in Sf-9 cells, probably homologs of mammalian Gi/Go proteins. Despite the apparent coupling of the D3 receptor to G-proteins, GTPgammaS (10 microM) failed to decrease agonist binding ([3H]dopamine) to the D3 receptor, probably due to small affinity differences between low and high affinity states for agonists in the D3 receptor, as well as due to high receptor density in Sf-9 cells. We conclude that agonist-induced [35S]GTPgammaS binding for the D3 receptor is suitable for estimating ligand intrinsic efficacy and pharmacological characterizations of ligand-receptor interactions.