Exogenous acetylcholine (ACh) is known to stimulate atrial natriuretic peptide (ANP) secretion concomitantly with a decrease in atrial pulse pressure. However, the role of intrinsic ACh in the regulation of ANP secretion remains unknown. Recently, it was shown that nonneuronal and neuronal ACh is present in the cardiac atria. From this finding we hypothesize that endogenously released ACh is involved in the regulation of ANP secretion in an autocrine or paracrine manner in the atria. Experiments were performed in isolated beating rat atria. ANP was measured using radioimmunoassay. To increase the availability of the ACh in the extracellular space of the atrium, its degradation was inhibited with an inhibitor of acetylcholinesterase. Acetylcholinesterase inhibition with physostigmine increased ANP secretion concomitantly with a decrease in atrial dynamics in a concentration-dependent manner. Inhibitors of M2 muscarinic ACh receptor (mAChR), methoctramine, and ACh-activated K(+) (KACh(+)) channels, tertiapin-Q, abolished the physostigmine-induced changes. The effects were not observed in the atria from rats treated with pertussis toxin. Furthermore, the physostigmine-induced effects were attenuated by an inhibitor of high-affinity choline transporter, hemicholinium-3, which is a rate-limiting step of ACh synthesis. Inhibitors of the mAChR signaling pathway and ACh synthesis also attenuated the basal levels of ANP secretion and accentuated atrial dynamics. These findings suggest that endogenously released ACh tonically stimulates ANP secretion from atrial cardiomyocytes via activation of M2 mAChR-Gi/o-KACh(+) channel signaling. It is also suggested that the ACh-ANP signaling is implicated in cardiac physiology and pathophysiology.