The ability of cloned human O6-methylguanine-DNA methyltransferase to repair a methylated guanine in a CpG-containing sequence, i.e., island, was studied by using a synthetic double-stranded 20-mer oligonucleotide from codon 248 of the p53 gene and another designed sequence. The double-stranded oligonucleotides incorporating 5-methylcytosine (5mC) and O6-methylguanine (O6mG) in various combinations in a CpG site were 5' labeled with 32P and incubated with recombinant O6-methylguanine-DNA methyltransferase. The rate constant for O6-methylguanine-DNA methyltransferase repair of O6mG in this oligomer was always higher with the substrate which contained only the O6mG, as compared to the oligomer that included a 5mC adjacent in the 5'-position to the methylated guanine. The reduction in substrate activity ranged from 75% (modified p53 sequence) to 100% (in the designed oligomer). A 5mC opposite the O6mG reduced the rate slightly. These results suggest that O6-methylation of the guanine moiety at CpG islands may not be efficiently repaired when normal 5mC is present and this may contribute significantly to an increase in mutagenesis of p53 and like molecules.