IFN-γ is a potent pleiotropic Th1 cytokine, the production of which is tightly regulated during fetal development. Negative control of fetal/neonatal IFN-γ production is generally attributed to the Th1-antagonistic effect of mediators produced by the placenta, but evidence exists of additional and more direct transcriptional regulation. We report that neonatal (cord blood) CD3+/CD45RO− T cells, in particular the CD4+/CD45RO− subset, are hypermethylated at CpG and non-CpG (CpA and CpT) sites within and adjacent to the IFN-γ promoter. In contrast, CpG methylation patterns in cord blood IFN-γ-producing CD8+/CD45RO− T cells and CD56+/CD16+/CD3− NK cells did not differ significantly from those in their adult counterparts. Consistent with this finding, IFN-γ production by stimulated naive cord blood CD4+ T cells is reduced 5-to 10-fold relative to adult CD4+ T cells, whereas production levels in neonatal and adult CD8+ T cells are of a similar order. Evidence of significant CpA and CpT methylation was not discovered in promoter sequence from other cytokines (IL-4, TNF-α, or IFN-γR α-chain). We additionally demonstrate that overexpression of DNA methyltransferase 3a in embryonic kidney carcinoma cells is accompanied by CpA methylation of the IFN-γ promoter.