The generation of killer cell Ig-like receptor (KIR) expression patterns in NK cells involves variegated silencing of KIR genes by DNA methylation. To identify regulatory elements involved in KIR gene activation, upstream regions of KIR genes were functionally characterized in NK3.3 cells as well as in primary NK cells. Three kinds of KIR promoters were defined, controlling clonally expressed KIR genes, the constitutively active KIR2DL4, and the weakly expressed KIR3DL3. Upstream of a short core promoter common to all KIR genes, a region containing functionally divergent elements was characterized. Although this region had no impact on the activity of the KIR2DL3 promoter, an inhibitory element was identified in the KIR2DL4 promoter and an activating element was found in the KIR3DL3 promoter. Upon treatment with a methyltransferase inhibitor, KIR3DL3 expression could be readily induced showing that the low levels of KIR3DL3 expression in peripheral blood are due to sustained DNA methylation of an otherwise fully functional promoter. Analysis of transcription factor binding sites identified a functional acute myeloid leukemia (AML) site common to all three KIR promoters. Mutation of this site led to a substantial increase in activity of all KIR promoters. Among the different members of the AML family, AML-2 was identified as the predominant KIR binding factor. The present study suggests that AML-2 acts as a repressor of KIR expression in mature NK cells and opens the possibility that AML factors and associated cofactors are involved in regulation of KIR expression during NK cell development.