Tetrahydrobiopterin (BH4) is an important cofactor for two hepatic enzymes, inducible nitric oxide synthase (iNOS) and phenylalanine hydroxylase (PAH), and competition for BH4 between the two enzymes might limit hepatic iNOS or PAH activity. To test this hypothesis, we determined whether conversion of phenylalanine to tyrosine was modified by changes in NO synthase activity, and conversely whether NO synthesis was limited by the rate of phenylalanine conversion to tyrosine in rat hepatocytes and perfused livers. NO production was decreased only slightly, when flux through PAH was maximized in isolated perfused livers, and in isolated hepatocytes only when BH4 synthesis was inhibited. Increases in NO synthesis did not reduce tyrosine formation from phenylalanine. Phenylalanine markedly increased biopterin synthesis, whereas arginine had no effect. Thus, basal BH4 synthesis appears to be adequate to support iNOS activity, whereas BH4 synthesis is increased to support PAH activity.