Acetylcholine (ACh) caused a dose-dependent contraction of gallbladder muscle cells in either a normal (1.9 mM) Ca2+, zero-Ca2+ or 4 mM Sr2+ medium, with a maximal contraction about 21 +/- 1% at 10(-6) M. Pirenzepine, methoctramine and p-fluoro-hexahydro-sila-difenidol (the M1, M2 and M3 antagonist, respectively) alone had no inhibitory effect on ACh-induced contraction in normal Ca2+ medium, which was blocked by the combination of methoctramine and p-F-HHSiD. In the 4 mM Sr2+ medium, methoctramine dose dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The contraction induced by ACh was further blocked by 10(-4) M propranolol (phosphatidic acid phosphohydrolase inhibitor that prevents the production of diacylglycerol from phospholipase D activation), 10(-5) M H-7 and chelerythrine (the protein kinase C inhibitors) by 64%, 75% and 77%, respectively. In contrast, in the zero-Ca2+ medium, p-fluoro-hexahydro-sila-difenidol dose-dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The action of ACh was further blocked by 10(-6) M U-73122 (phospholipase C inhibitor) and 10(-5) M CGS 9343B (calmodulin antagonist) by 95% and 77%, respectively. In conclusion, ACh contracts the gallbladder muscle by stimulating the M2 and M3 muscarinic receptors. The M2 receptors are linked to Ca2+ influx, activation of phospholipase D and protein kinase C-dependent pathway, whereas the M3 receptors are preferentially associated with the activation of phospholipase C, intracellular Ca2+ release and calmodulin-dependent pathway.