Prostaglar din H synthase (PHS), an arachidonic acid-dependent peroxidase, has been implicated in the peroxidative activation of carcinogenic aromatic amines in extrahepatic carcinogen target tissues of experimental animals. We have examined the arachidonic acid-dependent activation of [3H]benzidine to DNA-bound products by microsomal prearations from 75 normal human tissues obtained during necessary surgical procedures. For several samples of urinary bladder epithelium, prostatic epithelium, colonic mucosa, and peripheral lung tissue, an arachidonic acid-dependent, microsomal-catalyzed activation of benzidine was observed; and the activity could be inhibited appreciably by indomethacin, a known inhibitor of PHS. Little or no arachidonic acid-dependent activity was detected in human placenta, breast, or liver microsomes or the majority of colon microsomes. Substrate specificity was also examined with purified ram PHS and with human bladder and with active colon preparations. Purified PHS catalyzed the activation of benzidine ≫ 2-naphthylamine, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole > 4-aminobiphenyl > 2-amino-3-methylimidazo[4,5-f]quinoline > 3-amino-1-methyl-5H-pyrido[4,3-b] indole. In comparison, human bladder and colon microsomes catalyzed the activation of benzidine > 4-aminobiphenyl, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole, 2-naphthylamine > 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. To confirm the occurrence of PHS antigen in human extrahepatic tissues, an avidin/biotin-amplified competitive enzyme-linked immunoabsorbent assay was developed with purified ram PHS and a commercially available monoclonal antibody known to cross-react with human platelet PHS. The avidin/biotin-amplified enzyme-linked immunosorbent assay, which detected ng quantities of ram PHS, clearly established the presence of the PHS protein in human bladder, prostate, and lung microsomes. In contrast, PES antigen was not detected in the liver or placental microsomes. The interindividual and tissue-dependent variability of PHS and its role in aromatic amine carcinogenesis are discussed.