Benzo(a)pyrene was demonstrated to be an effective mutagenic agent in cultured human cells when it was subjected to metabolic activation by S-9 rat liver microsomes. Although dose-dependent response curves were nonlinear, a very strong direct relationship was demonstrated between mutation frequency at the HPRT locus and the formation of B(a)P-DNA adducts. Southern Blot analysis demonstrated that mutant cell lines often contained major chromosome alterations at the HPRT locus, either deletions or rearrangement. Among three restriction endonucleases evaluated, Hind 111 was most efficient at detecting DNA alterations. Spontaneous mutants and gamma-ray-induced mutants both displayed similar proportions of DNA alterations of various kinds, suggesting that mutation events are approximately random at this locus. All mutant cell lines which are determined to have major DNA alterations had no or very low HPRT enzyme activity. Those mutant lines which could not be demonstrated to have substantial rearrangements or deletions had low, but measurable, enzyme activity.