In this study, the ligand-receptor-G protein interactions of the dopamine D3 receptor expressed in Chinese hamster ovary cells were investigated using guanosine 5'-[gamma-thio]triphosphate-[35S] ([35S]GTPgammaS) and receptor binding experiments. Dopamine stimulated the [35S]GTPgammaS binding in a guanine nucleotide, magnesium and sodium-dependent manner. Dopamine and quinpirole produced maximal stimulation of the [35S]GTPgammaS binding whereas (+)-7-OH-DPAT and (-)-3-PPP were partial agonists. Interestingly, several compounds previously classified as D2 receptor antagonists behaved as inverse agonists at the D3 receptor, i.e., they inhibited the basal [35S]GTPgammaS binding in a dose dependent fashion. Haloperidol, (+)-UH-232, (+)-AJ-76 and raclopride were full inverse agonists but clozapine was a partial inverse agonist. Pertussis toxin treatment abolished the D3 receptor-mediated agonist as well as inverse agonist responses, indicating the involvement of Gi/Go proteins in both processes. According to the ternary complex model, agonists should bind with higher affinity to the G protein coupled receptor (RG) and thereby shift the equilibrium from free receptor (R) toward RG, which produces a biological response. However, an inverse agonist should bind with higher affinity to R than to RG and thereby inhibit the basal activity of the cell. We found that the high affinity agonist binding site (RG) was abolished by pertussis toxin treatment of the cells. However, the inverse agonists bound with the same affinity to untreated and pertussis toxin treated D3 receptor membranes. Thus, we found no evidence for the hypothesis that inverse agonists would shift the equilibrium from RG toward R by binding with higher affinity to R than to RG.