Acetylcholine (ACh) release slows heart rate and atrioventricular conduction by stimulation of an inward rectifying current (IK,ACh) in atrial tissue. The effect of ACh on ventricular function is still debated. We compared the effect of ACh on APs in canine atria, Purkinje and ventricular tissue as well as on ionic currents in isolated cells. Action potentials were recorded from endo- or epicardial slices, Purkinje fibers, or atrial preparations. Whole-cell currents were recorded under voltage clamp conditions and unloaded cell shortening determined by video edge detection. The effects of ACh (1-10 μM) on IK,ACh and ICa in the 4 cell types were measured. In atrial tissue, application of ACh hyperpolarized the membrane potential and shortened action potential duration (APD). In Purkinje and ventricular tissues, no significant effect of ACh on APD, membrane potential or dV/dt was observed at 1 Hz pacing. Under voltage clamp, addition of ACh to atrial cells activated a large inward rectifying current (from −3.5±0.7 to −23.7±4.7 pA/pF) that was abolished by tertiapin, a specific blocker of IK,ACh. No significant enhancement of this current was observed in the other cell types following ACh application. A small inhibition of ICa was observed in all cell types after ACh. This ICa inhibition became greater at fast pacing rates. In the canine heart, application of ACh resulted in a marked reduction in APD in atrial tissue only. The effect on APD could be inhibited by an IK,ACh blocker suggesting this channel type was predominantly expressed in atrial tissue. In epi, endo and Purkinje tissue, no significant effect of ACh on APD was observed. A minor rate-dependent effect of ACh on ICa was noted however, the effect of this finding may be negligible at physiologically relevant rates.