We describe a rapid and sensitive, monoclonal antibody (mAb)-based immunoassay that permits the quantitation of cellular enzymatic activity for repair of specific carcinogen-DNA adducts. The assay was established on the basis of the observation that complexes between an anti-(O6-ethyl-2'-deoxyguanosine) mAb (mAb ER-6) and O6-ethylguanine (O6-EtGua) residues in double-stranded DNA can be immobilized on nitrocellulose filters. Circular pSV2gpt plasmid DNA was linearized by digestion with EcoRI and reacted with N-ethyl-N-nitrosourea in vitro to a level of about one O6-EtGua residue per DNA molecule, labeled with 32P, and subsequently incubated with extracts prepared from L929 mouse fibroblasts and with mAb ER-6. The amount of repaired O6-EtGua is proportional to the decrease in 32P activity retained on the filters. Maximum O6-alkyl-guanine repair activity requires preparation of the cell extracts in the presence of high (approximately 0.5 M) NaCl concentrations, whereas the repair reaction per se is markedly inhibited by NaCl. Under the conditions used, the detection limit of the assay is approximately 1 x 10(-16) mol of repaired O6-alkylGua. The repair reaction followed second-order kinetics, indicating that O6-EtGua is predominantly repaired by an enzyme activity inactivated by the repair reaction (e.g. via an alkyl group transfer mechanism analogous to that exhibited by the alkyl group accepting cysteine residues of the 'suicidal' bacterial ogt and ada proteins). The data obtained from the assay can be used for computation of the relative enzymatic repair activity of a given cell extract and of the rate constant K (1 x mol-1 x s-1) of the repair reaction.