As part of the innate immune system, human NK cells play a critical role early in the systemic host defense against pathogens and tumor cells. Recent studies suggest a more complex view of NK cell behavior, as different functions and tissue localizing capabilities seem to be preferentially assigned to distinct subpopulations of NK cells, CD56 dim CD16+ or CD56 bright CD16−. In this study, we used oligonucleotide microarrays to compare the expression profile of∼ 20,000 genes in three NK cell subpopulations: peripheral blood-derived CD56 dim CD16+, CD56 bright CD16−, and in vitro-activated CD16+ NK cells. The differential expression of selected genes was verified by flow cytometry and functional assays. When comparing CD56 dim CD16+ and CD56 bright CD16− subsets, a new heterogeneous molecular basis for the functional and developmental differences between these two subsets was revealed. Furthermore, systematic analysis of transcriptional changes in activated CD16+ NK cells provided us with a better understanding of NK function in inflamed tissues. We highlight a number of genes that were overexpressed upon activation (eg, OX40 ligand, CD86, Tim3, galectins, etc.), that enable these cells to directly cross-talk with other innate and adaptive immune effectors. The overexpressed genes assign novel intriguing immunomodulatory functions to activated NK cells, in addition to their potent cytotoxic abilities.