Hepatocytes are capable of repeated inducible NO synthase (iNOS) expression, which occurs under inflammatory and stress conditions. This iNOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of iNOS in these cells, we characterized the iNOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of iNOS in cytokine-stimulated hepatocytes and that the iNOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released iNOS to the soluble fraction. iNOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted iNOS exhibits a lower specific activity than iNOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric iNOS without a greater relative distribution of iNOS to the peroxisomal pool. Thus, iNOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric iNOS. iNOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme.