We have recently shown that cyproterone acetate (CPA), an active component of some antiandrogenic drugs, induces the formation of DNA adducts detectable by the 32P-DNA postlabelling technique in rat liver. The postlabelling technique, however, does not provide evidence for the chemical nature of the adducts observed. To ascertain whether the CPA-induced adducts do contain CPA, we have incubated tritiated CPA with cultured hepatocytes from female rats, digested the DNA to 3'-monophosphonucleosides, extracted the DNA adducts formed into butanol and phosphorylated the adducts in the extract with unlabelled ATP. One major and one minor 3H-labelled adduct spot were detectable on the TLC chromatograms. The spots appeared at positions identical to those observed in the 32P-postlabelling experiments with unlabelled CPA. Furthermore, the ratio of 3H activity for the major versus the minor adduct spot was 11.9 +/- 1.8, which agreed with the corresponding ratio for the 32P activities, which was 13.2 +/- 3.5. These findings indicate that the CPA-induced DNA adducts, which we have previously detected by 32P-postlabelling do contain CPA or CPA metabolites.