Abstract The direct-acting mutagenicity of 1-nitropyrene (1-NP), a tumorigenic environmental pollutant and model nitropolycyclic aromatic hydrocarbon, was studied in Chinese hamster ovary (CHO) cells. Previous reports indicated that 1-NP, a potent direct-acting mutagen in Salmonella typhimurium, was mutagenic in CHO cells only in the presence of an exogenous activation system. In this study, a DNA-repair-deficient CHO cell line, CHO-UV5, and the repair-proficient CHO-K1-BH 4 cell line were used to measure the direct-acting mutagenicity of highly purified samples of 1-NP. Exposure of CHO-K1-BH 4 cells for 5, 12, and 24 hr did not increase mutations at the hypoxanthineguanine phosphoribosyl transferase ( hprt) locus ( p > 0.1; paired t test; n = 5–6), while treatment of CHO-UV5 cells for 24 hr produced a statistically significant induction of mutants (22 ± 6 mutants per 10 6 clonable cells vs a solvent control value of 9 ± 3 per 10 6 cells; p < 0.01; n = 6). 32P-Postlabeling analysis of the DNA adducts formed in CHO-UV5 cells following 1-NP exposure revealed the presence of a single major adduct. Based on its chromatographic properties, its sensitivity to nuclease P1 digestion, and its resistance to hydrazine treatment, the adduct appeared to be N-(deoxyguanosin-8-yl)-1-aminopyrene, which was presumably produced by nitroreduction of 1-NP to N-hydroxy-1-aminopyrene. These results suggest that the use of extended treatment times and DNA-repair-deficient cells may be required to assess adequately the mutagenicity of nitropolycyclic aromatic hydrocarbons in CHO cells.