The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M(2) receptor were studied on the L-type Ca(2+) currents (I(Ca,L)) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I(Ca,L) but did not significantly affect basal I(Ca,L). Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I(Ca,L) were not modified in presence of B8E5. Inhibition of I(Ca,L) by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase [1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one]. These results indicate that the antibody B8E5 inhibits the beta-adrenergic-stimulated I(Ca,L) through activation of the M(2) muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.