The role of rat O6-alkylguanine-DNA alkyltransferase (AGT) in modulating mutagenesis by O6-substituted guanines in the rat H-ras gene was examined. Rat4 cells were transfected with vectors carrying O6-methyl-, O6-ethyl- or O6-benzylguanine residues in place of the normal guanines at either the first, second, or both the first and second positions in codon 12 (GGA) of the H-ras coding sequence. The percentage of transformed colonies was determined for cells grown in normal medium or in medium containing O6-benzylguanine to completely deplete AGT. In parallel experiments with O6-methylguanine-containing vectors, the percentage of cellular DNA harboring codon 12 mutations was determined for normal cells and cells lacking AGT. A reasonable correspondence was observed between the percentage of mutated DNA and the percentage of transformed colonies produced in both types of cells. The results indicate that the contribution of AGT to the repair of O6-substituted guanine damage decreases as the O6 substituent is changed from methyl > ethyl > benzyl. Additionally, cellular AGT appears to repair an O6-methylguanine more readily at the first position of codon 12 than the second position. However, other repair mechanisms in these mammalian cells appear to play a major role in correcting low levels of O6-substituted guanine damage including O6-methylguanine damage.