Cardiac vagal control has prognostic significance in cardiac disease, but the control mechanisms of this system remain poorly understood. We have previously demonstrated a role for NO in promoting vagal control of heart rate in humans. Here we examine the influence of L-arginine, the substrate for NO synthase, on this mechanism in healthy human subjects. Eleven healthy volunteers (9 men; age, 20 to 25 years) underwent measurement of heart rate variability and baroreflex sensitivity before and during a systemic infusion of L-arginine (1 g/min; total, 30 g). To control for the fall in blood pressure, comparison was made with an infusion of the control vasodilator hydralazine. Stereospecificity of observed effects was investigated by infusion of D-arginine. Urinary nitrate and nitrite (NO(x)) and cGMP concentrations were measured as indexes of NO generation. L-Arginine infusion produced a drop in mean arterial pressure of 5 mm Hg. This fall in blood pressure was matched by hydralazine infusion and was not observed with either D-arginine or saline infusion. Although RR interval duration, heart rate variability, and baroreflex sensitivity all fell significantly with hydralazine, the same degree of baroreflex unloading with L-arginine produced an increase in RR interval duration and no change or even slight increases in heart rate variability and baroreflex sensitivity. In contrast, D-arginine produced falls in high-frequency indexes of heart rate variability compared with saline. Only L-arginine increased urinary NO(x) and cGMP excretion. In conclusion, these data demonstrate that short-term L-arginine infusion facilitates vagal control of heart rate in healthy humans, probably via increased NO synthesis.