To determine the potential role of methylation in the regulation of interferon-γ (IFN-γ) gene transcription by T cells, primary T-lineage cell populations were analyzed for the extent of methylation of three CpG sites within or near transcriptional activator elements in the 5′ flank and first intron of the human IFN-γ gene. A striking correlation was observed between the capacity of the IFN-γ gene to be expressed and the degree of hypomethylation. The IFN-γ gene was virtually completely methylated at all sites in thymocytes, neonatal T cells, and adult CD45RAhiCD45R0lo (antigenically naive) CD4 T cells, cell types that all have a low or undetectable capacity to express the IFN-γ gene. In contrast, there was substantial hypomethylation in T-lineage cell types with relatively high capacities to express the IFN-γ gene, including adult CD8 T cells and adult CD45RAloCD45R0hi (memory/effector) CD4 T cells. These results suggest that hypomethylation of the IFN-γ genetic locus may be an important determinant of IFN-γ gene expression in vivo by T-lineage cells.