Hyperthyroidism enhances the prooxidant activity of the liver by elevating superoxide radical and/or hydrogen peroxide generation in microsomal, mitochondrial, and peroxisomal fractions, with an increased respiratory burst of Kupffer cells. In this study, the influence of daily doses of 0.1 mg 3,3',5-triiodothyronine (T3)/kg for three consecutive days on liver nitric oxide (NO) synthase (NOS) was assessed, as a possible contributory mechanism to T3-induced liver prooxidant activity. Thyroid calorigenesis was paralleled by a progressive increment in the rate of NO generation, with significant increases after 2 (47%) and 3 days (70%) of T3 treatment, and a net 45% (P < 0.05) enhancement in the NG-methyl-L-arginine-sensitive NO production, compared to control values. These enhancement effects were reversed to control levels after 3 days of hormone withdrawal, concomitantly with the normalization of hepatic respiration. Enhancement of liver NOS activity in hyperthyroid animals was diminished by 27% (P < 0.05) by the selective in vivo inactivation of Kupffer cells by gadolinium chloride (GdCl3), without direct actions of GdCl3 on the enzyme. These data demonstrate that hyperthyroidism leads to a significant and reversible enhancement in rat liver NOS activity, an effect that is exerted at hepatocyte and Kupffer cell levels, thus representing an additional source of prooxidants to those of reactive oxygen species.