We have determined the mutational specificity of S9-activated benzo[a]pyrene (B[a]P) at the endogenous aprt locus in a hemizygous Chinese hamster ovary cell line. The aprt gene of recovered mutants was amplified using the polymerase chain reaction (PCR) and directly sequenced. This spectrum was then compared to mutations recovered following treatment with the B[a]P metabolite, benzo[a]pyrene diol-epoxide (BPDE). No significant difference between the two spectra in the types of mutations produced, or their distribution was observed. This observation supports the hypothesis that BPDE is the reactive metabolite of B[a]P, responsible for the significant biological effects caused by this ubiquitous polycyclic aromatic hydrocarbon. The major mutation recovered was the G:C-->T:A transversion, and mutations were primarily localized within runs of guanines. We also confirmed our previous finding that mutation by B[a]P is non-random, targeting events in runs of guanines flanked by adenine residues. This same target hotspot region is found in codon 61 of the human c-Ha-ras1 proto-oncogene. This may help explain the selective activation of this codon by BPDE.