Human NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the mechanisms that regulate DAP10 gene expression. We describe the existence of multiple transcriptional start sites upstream of DAP10 exon 1 and identify the location of the basic promoter upstream of these starting sites. The promoter is active in NK and CD8+ T cells, but not in CD4+ T cells. We demonstrate TCR-mediated up-regulation of DAP10 transcription and found that a 40 bp region within the DAP10 promoter, containing an Ap-1 binding site, is largely responsible for this increased transcription. Using pull-down and chromatin immunoprecipitation assays, we show that the DAP10 promoter interacts with Ap-1 transcription factors in primary CD8+ T and NK cells in vitro and in vivo. Overexpression of c-Jun or c-Fos in NK and T cells led to enhanced DAP10 promoter activity and DAP10 protein expression. Taken together, our data indicate that Ap-1 is an important transcription factor for regulating DAP10 gene expression in human NK and T cells, and that Ap-1 plays a key role in the transactivation of DAP10 promoter following TCR stimulation.