The major mutational hot spots in human cancers occur at CpG sequences in the p53 gene. It is generally presumed that the majority of mutations at these sites result from the endogenous deamination of methylated cytosine. Using a UvrABC incision method, we have found that cytosine methylation greatly enhances guanine alkylation at all CpG sites in the p53 gene by a variety of carcinogens, including benzo(a)pyrene diol epoxide, benzo(g)chrysene diol epoxide, aflatoxin B1 8,9-epoxide, and N-acetoxy-2-acetylaminofluorene. These findings suggest that mutational hot spots at methylated CpG sequences in the p53 gene may be a consequence of preferential carcinogen binding at these sites.