Dibenz(a,h)anthracene (DB(a,h)A) has been studied to identify the major routes of metabolic activation in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. The morphological transforming activities of three potential intermediates formed by metabolism of DB(a,h)A by C3H10T1/2 cells, trans-3,4-dihydroxy-3,4-dihydro-DBA (DBA-3,4-diol), anti-trans-3,4-dihydroxy-3,4-dihydro-DBA-1,2-oxide (DBA-3,4-diol-1,2-oxide), and DBA-5,6-oxide were determined. DBA-3,4-diol-1,2-oxide was a strong morphological transforming agent giving 73% dishes with Type II or III foci at 0.5 micrograms/ml. DBA-3,4-diol and DB(a,h)A had similar activities, approximately 24-42% dishes with Type II or III foci at 2.4 micrograms/ml. DBA-5,6-oxide, was found to be inactive. DNA adducts of DB(a,h)A, DBA-3,4-diol, DBA-3,4-diol-1,2-oxide, and DBA-5,6-oxide in C3H10T1/2 cells were isolated, separated, and quantitated using the (32)P-postlabeling method. Qualitatively, all of the DNA adducts observed in C3H10T1/2 cells treated with DB(a,h)A were also observed in the DNA of these cells treated with DBA-3,4-diol. These results indicate that DB(a,h)A is metabolically activated through DBA-3,4-diol in C3H10T1/2 cells. Of the DNA adducts formed, 86% are a result of the further metabolism of DBA-3,4-diol to DBA-3,4-diol-1,2-oxide. These studies provide little evidence for the metabolism of DB(a,h)A by the K-region pathway.