The formation and persistence of O6-ethylguanine, O4-ethylthymine, and O2-ethylthymine were quantitated in the genomic DNA of human lymphoblasts exposed to 1.0 mM N-ethyl-N-nitrosourea using immunoslot-blot. The three cell lines used included one which lacks O6-alkylguanine-DNA alkyltransferase, one deficient in nucleotide excision repair, and a third which is competent in both of these repair pathways. The activity of O6-alkylguanine-DNA alkyltransferase was further modulated with O6-benzylguanine, a specific inhibitor of this protein. Repair of the O-ethylated thymines was slow and not related to either DNA repair phenotype. O6-Ethylguanine was repaired with a half-life of about 8 h in cells which expressed both O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair functions. Cells expressing O6-alkylguanine-DNA alkyltransferase activity but lacking nucleotide excision repair showed only slow repair of O6-ethylguanine (half-life of O6-ethylguanine, 43 h), while cells lacking the alkyltransferase showed little or no repair of O6-ethylguanine regardless of nucleotide excision repair activity (half-lives of O6-ethylguanine, 53 to greater than 100 h). We conclude that O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair cooperate in the repair of O6-ethylguanine in human cells.