The polycyclic aromatic hydrocarbon naphthalene is an environmental pollutant, a component of jet fuel, and, since 2000, has been reclassified as a potential human carcinogen. Few studies of the in vitro human metabolism of naphthalene are available, and these focus primarily on lung metabolism. The current studies were performed to characterize naphthalene metabolism by human cytochromes P450. Naphthalene metabolites from pooled human liver microsomes (pHLMs) were trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol, and 2-naphthol. Metabolite production generated Km values of 23, 40, and 116 microM And Vmax values of 2860, 268, and 22 pmol/mg protein/min, respectively. P450 isoform screening of naphthalene metabolism identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production. Metabolism of the primary metabolites of naphthalene was also studied to identify secondary metabolites. Whereas 2-naphthol was readily metabolized by pHLMs to produce 2,6- and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were inefficient substrates for pHLMs. A series of human p450 isoforms was used to further explore the metabolism of dihydrodiol and 1-naphthol. 1,4-Naphthoquinone and four minor unknown metabolites from 1-naphthol were observed, and CYP1A2 and 2D6*1 were identified as the most active isoforms for the production of 1,4-naphthoquinone. Dihydrodiol was metabolized by P450 isoforms to three minor unidentified metabolites with CYP3A4 and CYP2A6 having the greatest activity toward this substrate. The metabolism of dihydrodiol by P450 isoforms was lower than that of 1-naphthol. These studies identify primary and secondary metabolites of naphthalene produced by pHLMs and P450 isoforms.