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, we estimated directly the acute effect of insulin on intravascular NOx fractional (FSR) and absolute (ASR) synthesis rates in vivo. Nine male healthy volunteers were infused iv with L-[¹⁵N₂-guanidino]-arginine for six hours. Timed measurements of ¹⁵NOx and ¹⁵N₂-arginine enrichments in whole blood were performed in the first 3 hours in the fasting state, thereafter following a 3-hr euglycemic-hyperinsulinemic clamp (with plasma insulin raised to about|1000 pmol/L). In the last 60 min of each experimental period, at steady-state arginine enrichment, a linear increase of ¹⁵NOx enrichment (mean r = 0.9) was detected in both experimental periods. In the fasting state, NOx FSR was 27.4±4.3 percent x day^-1, whereas ASR was 0.97±0.36 mmol x day^-1, accounting for 0.69 ±0.27% of arginine flux. Following hyperinsulinemia, both FSR and ASR of NOx increased (FSR: by 50%, to 42.4 ±6.7 percent x day^-1, p<0.005; ASR: by 25%, to 1.22 ±0.41 mmol x day^-1, p=0.002), despite a 20-30% decrease of arginine flux and concentration. The fraction of arginine flux used for NOx synthesis was doubled, to 1.13 ±0.35% (p<0.003). In conclusion, whole-body NOx synthesis can be directly measured over a short observation time with stable isotope methods in humans. Insulin acutely stimulates NOx synthesis from arginine.