The mutagenic specificity of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline (MeIQx), a food-borne mutagen and carcinogen, was studied. Plasmid pK19 was modified by photolysis with the 2-azido form of the carcinogen. High pressure liquid chromatography confirmed that the photoactivated azide formed primarily C8 and N2 guanyl adducts. Transformation of modified pK19 into excision repair competent Escherichia coli resulted in dose-dependent increases in genotoxicity and in mutagenesis within the lacZalpha target sequence. Upon induction of the SOS response, a 20-fold increase in mutation frequency over background was observed. A mutational spectrum for MeIQx, generated by sequencing 125 independent mutants, revealed base substitutions (41%), frameshifts (54%), and complex mutations (5.6%); >90% of the mutations occurred at G-C base pairs. Two hotspots were evident at runs of three or five G-C base pairs; approximately 60% of the mutations occurred at the hotspot sites. The hotspot at position 2532 produced mainly base substitutions, while that at position 2576 gave exclusively frameshift mutations. A polymerase inhibition assay mapped the sites of MeIQx adducts. Arrest sites were primarily at or one base 3' to a guanine residue, which correlated well with the distribution of mutations. No direct correlation was seen, however, between intensity of modification and hotspots for mutation.