We studied the functions of subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^- currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^- channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE) and baclofen enhanced the ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor, -opioid receptor or GABA_B receptor, respectively. 5-HT also enhanced the pituitary adenylate cyclase activating peptide 38 (PACAP38)-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor-CFTR and 5-HT_1A receptor. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of G-activated form of adenylate cyclase (AC) type II, but not AC type III. Thus, subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1A receptor or -opioid receptor alone, but elicited Ca²⁺-activated Cl^- currents in oocytes coexpressing these receptors with G -activated form of phospholipase C (PLC)-2, but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses and intracellular Ca²⁺ mobilization.