As determined by a guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, which does not distinguish G protein subtypes, 5-hydroxytryptamine (5-HT) and 2(S)- 1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) behaved as full agonists at human 5-HT(2C) (h5-HT(2C)) receptors (VSV isoform) stably expressed in Chinese hamster ovary (CHO) cells, whereas 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), d-lysergic acid diethylamide (LSD), and lisuride exhibited partial agonist properties. After treatment with pertussis toxin to uncouple 5-HT(2C) receptors from Gi/Go but not Gq/11, DOI and LSD were as efficacious as 5-HT and Ro600175 in stimulating [(35)S]GTPgammaS binding, whereas lisuride still exhibited low efficacy (40%). Correspondingly, in a scintillation proximity assay employing specific antibodies against Gq/11, 5-HT, Ro600175, DOI, and LSD behaved as high-efficacy agonists, whereas lisuride showed efficacy of 36%. In contrast, when employing a specific antibody recognizing Gi(3), DOI and LSD were less efficacious (80 and 30%, respectively) than 5-HT and Ro600175, and lisuride was inactive. Agonist actions were specifically mediated by h5-HT(2C) receptors inasmuch as the selective 5-HT(2C) antagonist SB242,084 blocked [(35)S]GTPgammaS binding at both Gq/11 and Gi(3). Agonist potency for stimulation of Gi(3) was ~6- to 8-fold less than for Gq/11, indicating that the latter was preferentially engaged by h5-HT(2C) receptors. Inactivation of h5-HT(2C) receptors with the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline did not modify the efficacy of 5-HT, Ro600175, and DOI at Gq/11, whereas their efficacies were substantially reduced at Gi(3), indicating a greater receptor reserve for the former. Finally, the preferential activation of Gq/11 versus Gi(3) by DOI, LSD, and lisuride was diminished in the presence of lower receptor number. In conclusion, h5-HT(2C) receptors couple to both Gq/11 and Gi(3) in CHO cells, and efficacy for G protein subtype activation is both ligand- and receptor reserve-dependent.