The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4(+) T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19(+) B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4(+) T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4(+) T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4(+) T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4(+) cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosuppression.