Protoporphyrin IX inhibits citrulline formation by all three nitric oxide synthase isoforms in a manner reversible by dilution. Zinc protoporphyrin IX, by contrast, produces a time- and concentration-dependent inactivation of all three nitric oxide synthase isoforms, not reversible by dilution. The inhibition of citrulline formation by protoporphyrin IX occurs with IC₅₀values of 0.8, 4, and 5 μfor the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition byN-methyl-protoporphyrin IX occurs at IC₅₀values of 6, 5, and 8 μfor the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition of nitric oxide synthase by protoporphyrin IX is a multisite, positively cooperative inhibition that exhibits a Hill coefficient of 2.3 for the iNOS isoform. Protoporphyrin IX reduces the maximal velocity of citrulline formation for both the iNOS and nNOS isoforms without altering theK_mfor the arginine substrate or the EC₅₀value for the tetrahydrobiopterin cofactor. Protoporphyrin IX inhibits the arginine-independent NADPH oxidase activity of nNOS with an IC₅₀value of 1 μbut has no effect on cytochrome c reductase activity at concentrations as high as 30 μ. At concentrations of 10 and 20 μ, protoporphyrin IX inhibits NO formation by cytokine-induced murine RAW 264.7 cells; however, these inhibitions are accompanied by significant cellular cytotoxicity. Coproporphyrins I and III, uroporphyrins I and III, and porphobilinogen, intermediates in the biosynthesis of heme that accumulate in hepatic porphyrias, are ineffective as inhibitors of the nitric oxide synthase isoforms. Since protoporphyrin IX is the immediate biosynthetic precursor of heme that accumulates in hepatic protoporphyria, iron deficiency anemia, and lead poisoning, protoporphyrin IX inhibition of nitric oxide synthase may contribute to the pathophysiology of these conditions.