The possible role of type II (cGMP-stimulated cAMP hydrolysis) phosphodiesterase (PDE) in the accentuated antagonism of muscarinic effects on heart rate during -stimulation via endogenous nitric oxide (NO) was evaluated. The canine isolated sinoatrial node preparation was cross circulated with arterial blood of a support dog. The sinoatrial rate of the preparation was 96 ± 5 beats/min (n = 16) at control. Methacholine (MCh; 0.01-1 µg) injected into the right coronary artery in a bolus fashion caused dose-dependent decreases in sinoatrial rate. Under an intra-arterial infusion of isoproterenol (1 µM), resulting in ~50% increase in sinoatrial rate, MCh-induced decreases were markedly augmented from 18 ± 3% to 44 ± 4% at 0.3 mg of MCh. When N^G-nitro-L-arginine methyl ester (100 µM) or N^G-monomethyl-L-arginine (100 µM) were continuously infused, the augmented MCh-induced decreases in sinoatrial rate were significantly suppressed (29 ± 3% or 25 ± 3%, respectively, P < 0.01). Pretreatment with either 3-isobutyl-1-methylxanthine (IBMX; 20 µM), a non-selective PDE inhibitor, or amrinone (20 µM), a selective type III (cGMP inhibited cAMP hydrolysis) PDE inhibitor, doubled the isoproterenol-induced increase in the sinoatrial rate. However, the augmented MCh-induced decreases in sinoatrial rate were significantly depressed by IBMX (from 23 ± 5% to 14 ± 1%, P < 0.01) but not by amrinone (to 20 ± 3%). These results suggest that MCh-induced accentuated antagonism in the sinoatrial node pacemaker activity can be modulated by endogenous NO via an activation of the type II cyclic GMP-stimulated cAMP PDE.