Pilocarpine, a nonselective agonist of the muscarinic cholinoreceptors, was shown to activate both M2 and M3 cholinoreceptors in atria under the conditions of microelectrode registration of action potentials in a rat heart. Activation of M3 receptors was demonstrated after selective blocking of M2 receptors with methoctramine (100 nM). This was accompanied by changes in the duration of the action potential and configuration of repolarization phases. The effect was removed by the 4-DAMP selective M3 receptor blocker (10 nM) and also by the inhibition of phospholipase C by U-73122 (1 μM). This suggests that the effects of M3 receptor activation in the atrium are associated, at least partially, with phosphoinositide signaling.