The probable human carcinogen 4,4'-methylene-bis(2-chloroaniline) (MOCA) was utilized to develop biomarkers of exposure to occupational carcinogens. The 32P postlabeling assay, utilizing the nuclease P1 enhancement procedure, was used to evaluate MOCA-DNA adduct formation in target tissues. Male Sprague-Dawley rats were treated with different dosing regimens of MOCA, and DNA was isolated from the liver. Additionally, a human uroepithelial cell (HUC) line was treated with N-hydroxy-MOCA for 24 hr, cells were harvested, and DNA was isolated. DNA was analyzed for MOCA-DNA adduct formation by the 32P postlabeling assay. Five MOCA adducts were detected in rat liver DNA. Adduct A, which corresponded to N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was the major adduct in rat liver DNA appearing in all treatment groups. Levels of adduct A were higher when MOCA was administered by ip injection versus oral gavage. Phenobarbital pretreatment increased the amount of adduct A approximately 12-fold. The pathway leading to the formation of adduct A in DNA from HUC appeared to be saturated at the concentrations used: 2.5, 5, and 10 microM. However, an additional adduct (E) was observed at the 10 microM treatment level only. A major DNA adduct was detected in the target tissue of rats and target human cells for MOCA-induced carcinogenesis, thus making it useful as a biomarker of exposure. Other DNA adducts were also observed with the different doses and routes of exposure investigated.