Mammary and skin tumors induced in rodents by N-methyl-N-nitro-sourea treatment have a G:C to A:T transition mutation in codon 12 of H-ras, probably resulting from alkylation of O6 of guanine by the carcinogen. This codon contains two guanines (5′-GGA-3′), but mutations are observed only in the central base pair of this codon. The same selectivity for mutations of -GGA-sequences has also been observed in Escherichia coli. It is known that the central G in the sequence GGA is a preferred site for alkylation, but the magnitude of chemical selectivity is insufficient to provide a complete explanation for the biological observation which is still unexplained. We have measured accurate rates of repair by the E. coli ada gene O6-alkylguanine-DNA-alkyltransferase of an O6-methylguanine in various positions in chemically synthesized 15-base pair DNA duplexes having the H-ras sequence. The rate of repair varied 25-fold, depending on the sequence flanking the methylguanine. An O6-methylguanine in position 2 of codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation for the selective mutation of this position. Using an antibody to probe the accessibility of the O6-methyldeoxyguanosine, it was shown that the rate of repair is a reflection of the conformation of the sequence containing the alkylated base, because the avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the methylguanine, with the most rapidly repaired O6-methylguanines being those most easily bound by the antibody.