Cytokine secretion profiles of activated T cells are critical for maintaining the immunologic balance between protection and tolerance. In mice, several cytokines have been reported to exhibit monoallelic expression. Previously, we found that the human interleukin-1 alpha (IL1A) gene exhibits a stable allele-specific expression pattern in CD4+ T-cell clones. We investigated whether DNA methylation is involved in the allele-specific expression of IL-1α. Here, we show that differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1α in CD4+ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1α, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of nonexpressing cells and of T-cell clones with 5-aza-2′deoxycytidine induced IL-1α expression in the nonexpressing cells and induced expression of the formerly silent allele in T-cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1α in CD4+ cells is achieved, at least in part, by differential methylation of the promoter.