The purpose of this study was to investigate the role of nitric oxide (NO) in mediating vagal and sympathetic modulation of spontaneous sinus cycle length (SCL) and atrioventricular (AV) nodal conduction time (A-H interval) in 62 open-chest mongrel dogs anesthetized with alpha-chloralose. Infusion of an NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA, 4 mg/ ml), into the sinus and AV nodal arteries attenuated significantly (P < 0.01) the negative chronotropic and dromotropic responses to vagal nerve stimulation (VS) and VS during ansae subclaviae stimulation (SS) or isoproterenol (Iso) infusion. Intravenous administration of L-arginine (100 mg/kg) reversed these responses toward control values, whereas D-arginine did not have a significant effect. L-NMMA significantly (P < 0.01) enhanced the effects of SS and Iso on SCL and A-H interval; L-arginine reversed these changes toward baseline. L-NMMA increased the minimum concentration of ACh needed to induce 50 or 100% prolongation of SCL or second-degree or complete AV block during concomitant Iso infusion. L-Arginine reversed these effects. NOS inhibition did not affect the direct cholinergic actions of ACh on SCL and A-H interval but enhanced adrenergic positive chronotropic and dromotropic effects. We conclude that NO plays a stimulatory role in mediating vagal neurotransmission and vagal modulation of sympathetic effects and an inhibitory role in mediating sympathetic neurotransmission.