A senescent CD4(+)CD28(-) T cell subset develops with aging and in chronic inflammatory diseases like rheumatoid arthritis, and is implicated in plaque rupture and myocardial infarctions. This subset is pro-inflammatory, cytotoxic for endothelial cells, and aberrantly expresses genes like CD70, perforin and killer cell immunoglobulin-like receptor (KIR) genes. Why CD4(+)CD28(-) cells overexpress these genes is unclear. We found that the CD70, perforin and KIR2DL4 promoters are demethylated in CD4(+)CD28(-) T cells, and that DNA methyltransferase 1 (Dnmt1) and Dnmt3a levels are decreased in this subset. siRNA "knockdown" of Dnmt1, but not Dnmt3a, in CD4(+)CD28(+) T cells caused similar demethylation and overexpression of KIR2DL4, perforin and CD70, while simultaneous knockdown of Dnmt1 and Dnmt3a caused greater demethylation and overexpression of these genes than Dnmt1 alone. We conclude that decreased Dnmt1 and Dnmt3a cause demethylation and overexpression of these and perhaps other genes in CD4(+)CD28(-) cells, potentially contributing to pathologic functions by this subset.