The in vitro metabolism of fluoranthene (FA) was assessed by incubating 3-[3H]FA, the synthesis of which is described, with rat hepatic microsomal enzymes. Several metabolites including the FA 2,3-diol, FA 2-3,-quinone, 3-OH-FA, 1-OH-FA, and 8-OH-FA were isolated by high-pressure liquid chromatography and identified by comparison of chromatographic properties and uv-visible spectra with those of synthetic standards. The major metabolite produced over the FA concentration range studied (23-233 microM) was FA 2,3-diol, accounting for 29-43% of the total extractable metabolites. This diol was characterized further by high-resolution mass spectroscopy and H-NMR and determined to be identical in structure to the trans-2,3-dihydroxy-2,3-dihydrofluoranthene. The FA 2,3-diol, syn and anti 2,3-diol-1,10b-epoxides, FA 2,3-quinone, and FA 7,8-diol were all shown to be mutagenic toward Salmonella typhimurium TM677. The FA 1,10b-diol and syn and anti FA 1,10b-diol-2,3-epoxides were not mutagenic. The epoxide hydrolase inhibitor, 3,3,3-trichloropropylene oxide, markedly reduced the mutagenic potency of FA while concurrently inhibiting FA 2,3-diol production but not overall FA metabolism. These results suggests that a major metabolic activation pathway of FA resulting in the production of mutagenic species involves the formation of the FA 2,3-diol and the subsequent oxidation of this diol to a FA 2,3-diol-1,10b-epoxide. Another minor activation pathway with mutagenic endpoints may involve the formation of the 7,8-diol.