IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8(+) T cells expressing IL-7Ralpha(high) and IL-7Ralpha(low) with different cell survival responses to IL-7. Although these CD8(+) T cell subsets have differential IL-7Ralpha gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Ralpha gene expression in human CD8(+) T cells and Jurkat T cells. IL-7Ralpha(high)CD8(+) T cells had decreased methylation in the IL-7Ralpha gene promoter compared with IL-7Ralpha(low)CD8(+) T cells and Jurkat T cells with low levels of IL-7Ralpha. Treating Jurkat T cells with 5-aza-2'-deoxycytidine, which reduced DNA methylation, increased IL-7Ralpha expression. Plus, the unmethylated IL-7Ralpha gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Ralpha expression in T cells via affecting IL-7Ralpha gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.