Nitric oxide (NO) is a key regulatory molecule with wide vascular, cellular, and metabolic effects. Insulin affects NO synthesis in vitro. No data exist on the acute effect of insulin on NO kinetics in vivo. By employing a precursor-product tracer method in humans, we have directly estimated the acute effect of insulin on intravascular NO(x) (i.e., the NO oxidation products) fractional (FSR) and absolute (ASR) synthesis rates in vivo. Nine healthy male volunteers were infused iv with L-[(15)N(2)-guanidino]arginine ([(15)N(2)]arginine) for 6 h. Timed measurements of (15)NO(x) and [(15)N(2)]arginine enrichments in whole blood were performed in the first 3 h in the fasting state and then following a 3-h euglycemic-hyperinsulinemic clamp (with plasma insulin raised to approximately 1,000 pmol/l). In the last 60 min of each experimental period, at approximately steady-state arginine enrichment, a linear increase of (15)NO(x) enrichment (mean r = 0.9) was detected in both experimental periods. In the fasting state, NO(x) FSR was 27.4 +/- 4.3%/day, whereas ASR was 0.97 +/- 0.36 mmol/day, accounting for 0.69 +/- 0.27% of arginine flux. Following hyperinsulinemia, both FSR and ASR of NO(x) increased (FSR by approximately 50%, to 42.4 +/- 6.7%/day, P < 0.005; ASR by approximately 25%, to 1.22 +/- 0.41 mmol/day, P = 0.002), despite a approximately 20-30% decrease of arginine flux and concentration. The fraction of arginine flux used for NO(x) synthesis was doubled, to 1.13 +/- 0.35% (P < 0.003). In conclusion, whole body NO(x) synthesis can be directly measured over a short observation time with stable isotope methods in humans. Insulin acutely stimulates NO(x) synthesis from arginine.