We have previously reported that feeding rats with a diet containing 0.02% L-N omega nitroarginine (L-NNA), a specific inhibitor of nitric oxide synthase, induced hypercholesterolemia. This present study was conducted to examine the underlying mechanism for hypercholesterolemia in rats. In experiment 1, feeding a diet containing 0.02% L-NNA for 5 wk elevated the concentration of serum cholesterol and reduced the excretion of fecal bile acids, but did not affect the excretion of fecal neutral sterols. Reduced activity of hepatic cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for the biosynthesis of bile acids from cholesterol, was observed in the rats receiving L-NNA. In experiment 2, rats were fed for 5 wk on a diet with or without 0.02% L-NNA that was or was not supplemented with 4% L-arginine. The L-NNA treatment elevated the serum concentrations of total cholesterol, free cholesterol and esterified cholesterol, and reduced the activity of hepatic cholesterol 7 alpha-hydroxylase, serum nitrate (a metabolite of NO) and the ratio of HDL-cholesterol versus serum total cholesterol. These alterations were suppressed by supplementing the L-NNA-containing diet with L-arginine. The results suggest that lower NO production by L-NNA caused hypercholesterolemia by a mechanism involving impaired bile acid synthesis.