Acetylcholine decreases currents through cardiac L-type Ca2+ channels after stimulation with agents which elevate levels of cyclic adenosine monophosphate, such as isoproterenol, but there is still a controversy over the mechanisms of this muscarinic effect. We tested the hypothesis of whether, after isoproterenol stimulation, protein phosphatases are activated by acetylcholine. Whole-cell currents were recorded from guinea-pig ventricular myocytes. The effect of 10(-5) M acetylcholine on currents induced by 10(-8) M isoproterenol was studied in the absence or presence of protein phosphatase inhibitors. Three agents reduced the acetylcholine response: okadaic acid (3 or 9 x 10(-6) M) and cantharidin (3 x 10(-6) M) added to the pipette solution, and bath-applied fluoride (3 mM). In contrast, pipette application of other phosphatase inhibitors, namely the inhibitor PPI2 (1000 U/ml), ciclosporin (10(-5) M), or calyculin A (10(-6) M) did not significantly diminish the acetylcholine effect. Interestingly, there was no correlation between the effects of the compounds on basal Ca2+ current and their interference with the muscarinic response. An activation of type 2A phosphatases by acetylcholine would explain these findings. Indeed, okadaic acid is 3 orders of magnitude more potent in vitro in its inhibition of this isoform (purified from cardiac myocytes) than is calyculin A, while type-1 phosphatases are inhibited equally. The data support the attractive possibility that stimulation of protein phosphatases is part of the signal transduction cascade of Ca2+ channel inhibition by acetylcholine.