We examined the role of endogenous NO in the autonomic regulation of atrioventricular (AV) nodal function by studying spontaneous action potentials (SAPs) and L-type Ca2+ current (ICa-L) in isolated single AV nodal cells from adult rabbit hearts. Both the perforated and the membrane-ruptured patch-clamp techniques in the whole-cell configuration were used under conditions known to alter NO production. Three NO donors, 3-morpholinosydnonimine (SIN-1, 0.1 mmol/L), S-nitroso-acetylcysteine (0.1 mmol/L), and sodium nitroprusside (0.1 mmol/L), suppressed the beta-adrenergic agonist isoproterenol (ISO, 1 mumol/L)-stimulated increase in ICa-L. SIN-1 also decreased the frequency and amplitude of SAPs. In cells in which ICa-L had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 mumol/L), SIN-1 had no additive effect. CCh activated an acetylcholine-sensitive outward K+ current (IK(ACh)) in AV nodal cells, in addition to the ICa-L inhibition. Intracellular dialysis with the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA, 0.5 mmol/L) blocked CCh-induced, but not SIN-1-induced, ICa.L attenuation. However, intracellular dialysis with methylene blue (20 mumol/L), which inhibits NO-mediated activation of guanylyl cyclase and cGMP production, blocked the effects of both CCh and SIN-1 on ICa-L. In these cells, neither L-NMMA nor methylene blue affected the CCh-activated IK(ACh). Direct application of cGMP (10 mumol/L) via internal dialysis significantly inhibited ISO-stimulated ICa-L. In AV nodal cells internally perfused with either a nonhydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit, ICa-L but still activated IK(ACh). CCh-induced ICa-L attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (20 mumol/L). However, CCh still significantly suppressed ISO-stimulated ICa-L after the cGMP-inhibited PDE isozyme (PDE3) had been selectively inhibited by milrinone (5 mumol/L). Immunohistochemical staining identified the presence of the endothelial constitutive NO synthase (ecNOS or NOS3) in both single AV nodal cells in vitro and in cryostat sections of AV nodal tissue in situ. These results demonstrate that endogenous NO is involved in the muscarinic cholinergic attenuation of ICa-L in AV nodal cell; the mechanism likely involves the cGMP-stimulated PDE.