Among the three G-protein-linked acetylcholine receptors (GARs) in Caenorhabditis elegans (C. elegans), GAR-3 is structurally and pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). Using Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the major alternatively spliced isoform of GAR-3, we observed that carbachol stimulated cyclic AMP (cAMP) production in a dose- and time-dependent manner. The stimulating effect of carbachol was abolished by atropine, a muscarinic antagonist, indicating that the cAMP production is specifically mediated by GAR-3b. When the cells were treated with BAPTA-AM and EGTA, which reduce the cytosolic Ca(2+) level, carbachol-stimulated cAMP accumulation was inhibited by approximately 56%. Inhibition of protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate (PMA) or by GF109203X decreased carbachol-stimulated cAMP production by as much as 68%. It thus appears that Ca(2+) and PKC are critically involved in GAR-3b-mediated cAMP formation. We also observed that carbachol-stimulated cAMP production was further enhanced by pertussis toxin (PTX) treatment. This observation indicates that GAR-3b couples to a PTX-sensitive G protein, presumably Gi, to attenuate the cAMP accumulation. Taken together, our data show that GAR-3b stimulates cAMP production in CHO cells and suggest that GAR-3b couples to both stimulatory and inhibitory pathways to modulate the intracellular cAMP level.