Genetic toxicology assays that rely on $9 microsomal mixes are subject to artifacts related to the generation of mutagenic metabolites by acidic p Hs, variation in individual isolations of microsomes and the failure of subcellular fractions to faithfully produce metabolites generated in intact cells. We have developed a gene mutation assay utilizing the human hepatoma cell line HepG2, which has been shown to metabolize a broad spectrum of promutagens. Optimal conditions for assaying the induction of 6-thioguanineresistant mutants in this cell line include: 1)growth of colonies for three weeks on lethally irradiated feeder layers of 106 thioguanineresistant Hep G2 cells (average plating efficiency = 60-80%); 2) a thioguanine concentration in selection dishes of l O-4 M with a maximum seeding density of 2.5 )< 105 cells per 100 mm culture dish; and 3) a minimum expression time of 6 days. In addition to ultraviolet light C (254 nm), a cytochrome t'45o (cyclophosphamide)dependent and a cytochrome P44s (aflatoxin BO-dependent promutagen were shown to induce cytotoxicity and mutations in this test system. The present studies, therefore, suggest that the HepG2 cell line may be useful for a variety of assays in genetic toxicology.