Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea. O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens. 