The purpose of the present study was to investigate whether the reversal by carbachol of positive inotropic responses of left atria to forskolin and isobutylmethylzanthine (IBMX) is related to the ability of carbachol to open potassium channels. Pretreatment of rabbits with pertussis toxin resulted in complete loss of the ability of carbachol to inhibit isoproterenol-stimulated adenylate cyclase activity and cyclic AMP (cAMP) generation in the left atria. Under these circumstances, negative inotropic responses of the left atria to carbachol in the presence of forskolin and IBMX were only partially attenuated, suggesting that at least part of the reversal by carbachol of the positive inotropic responses to forskolin and IBMX occurs by a cAMP-independent mechanism. The same dose of pertussis toxin had a much greater inhibitory effect on the ability of carbachol to reverse the isoproterenol-stimulated positive inotropic response. The potassium channel blocker, 4-aminopyridine, had no effect on the ability of carbachol to inhibit isoproterenol-induced increases in cAMP levels in atria from saline-treated rabbits. 4-Aminopyridine produced a concentration-dependent attenuation of the inhibitory effect of carbachol on positive inotropic responses to forskolin and IBMX in the left atria from both saline and pertussis toxin pretreated rabbits. These results suggest that the ability of carbachol to open potassium channels contributes to the cAMP-independent component of the negative inotropic response to carbachol in the presence of forskolin and IBMX.