Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated that high-affinity specific muscarinic receptors coupled to the phosphoinositide system are present in these cells. To determine whether muscarinic receptors in MDCK cells are linked negatively to the adenylate cyclase system, we measured the effect of muscarinic agonists and antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at 10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1, respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and forskolin were blocked by arecoline, a cholinergic agonist, in a concentration-dependent manner. The arecoline response was blocked by treatment of the cells with pertussis toxin. The inhibition by arecoline of forskolin-stimulated cAMP formation was reversed by various muscarinic antagonists in the following order of potency: 4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol > pirenzepine > methoctramine. This order of potency of muscarinic antagonists is similar to that observed in our radioligand binding studies and is consistent with the M3 subtype of muscarinic receptors. Our results indicate that muscarinic receptors in MDCK cells are coupled negatively to the adenylate cyclase system via pertussis toxin-sensitive G protein. It is concluded that this intracellular system may at least be partially responsible for the action of cholinergic agonists in these cells and in the kidney.