Folate is essential for the de novo biosynthesis of purines and thymidylate, and is an important mediator in the transfer of methyl groups for DNA methylation. Folate deficiency, therefore, could contribute to abnormal DNA integrity and methylation patterns. We investigated the effect of isolated folate deficiency in rats on DNA methylation and DNA strand breaks both at the genomic level and within specific sequences of the p53 tumor suppressor gene. Our data indicate that folate deficiency induces DNA strand breaks and hypomethylation within the p53 gene. Such alterations either did not occur or were chronologically delayed when examined on a genome-wide basis, indicating some selectivity for the exons examined within the p53 gene. Folate insufficiency has been implicated in the development of several human and experimental cancers, and aberrations within these regions of the p53 gene that were examined in this study are thought to play an integral role in carcinogenesis. The aforementioned molecular alterations may therefore be a means by which dietary folate deficiency enhances carcinogenesis.