The most prevalent DNA lesion induced by UV irradiation is the cyclobutane pyrimidine dimer (CPD), which forms at positions of neighboring pyrimidines. Here we show that the rare DNA base 5-methylcytosine is the preferred target for CPD formation when cells are irradiated with natural sunlight. We have mapped the distribution of CPDs formed in normal human keratinocytes along exons of the p53 gene. Codons 196, 245, 248, and 282, which are mutational hot spots in skin cancers, are only weakly to moderately susceptible to formation of CPDs after irradiation with UVC (254 nm) or UVB (320 nm) light sources. However, when cells were exposed to natural sunlight, CPD formation was enhanced up to 15-fold at these codons due to the presence of 5-methylcytosine bases. These results suggest that CPDs containing 5-methylcytosine may play an important role in formation of sunlight-induced skin tumors and that methylation of CpG sequences, besides being involved in spontaneous mutagenesis processes, can also create preferential targets for environmental mutagens and carcinogens.