Carcinogenic aromatic amines such as 4‐aminobiphenyl (ABP) and 2‐aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N‐hydroxylation catalyzed by CYP1A2 followed by O‐acetylation catalyzed by arylamine N‐acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP‐ and AF‐induced mutagenesis and DNA damage, nucleotide excision repair‐deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP‐ and AF‐induced hprt mutant cDNAs were sequenced and over 80% of the single‐base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in‐cell western assays and by identification and quantification of the two predominant DNA adducts, N‐(deoxyguanosin‐8‐yl)‐4‐aminobiphenyl (dG‐C8‐ABP) and N‐(deoxyguanosin‐8‐yl)‐2‐aminofluorene (dG‐C8‐AF) by liquid chromatography‐mass spectrometry. DNA damage and adduct levels were dose‐dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O‐acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine‐induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235–245, 2020. © 2019 Wiley Periodicals, Inc.