Mature human NK lymphocytes express the highly homologous killer Ig-like receptor (KIR) genes in a stochastic fashion, and KIR transcription precisely correlates with allele-specific DNA methylation. In this study, we demonstrate that CpG methylation of a minimal KIR promoter inhibited transcription. In human peripheral blood NK cells and long-term cell lines, expressed KIR genes were associated with a moderate level of acetylated histone H3 and H4 and trimethylated histone H3 lysine 4. Histone modifications were preferentially associated with the transcribed allele in NK cell lines with monoallelic KIR expression. Although reduced, a substantial amount of histone acetylation and H3 lysine 4 trimethylation also was associated with nonexpressed KIR genes. DNA hypomethylation correlated with increased chromatin accessibility, both in vitro and in vivo. Treatment of NK cell lines and developing NK cells with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, caused a dramatic increase in KIR RNA and protein expression, but little change in histone modification. Our findings suggest that KIR transcription is primarily controlled by DNA methylation.