Human natural killer (NK) cells can be subdivided in several subpopulations on the basis of the relative expression of the adhesion molecule CD56 and the activating receptor CD16. Whereas blood CD56CD16 NK cells are classically viewed as immature precursors and cytokine producers, the larger CD56CD16 subset is considered as the most cytotoxic one. In peripheral blood of healthy donors, we noticed the existence of a population of CD56CD16 NK cells that was frequently higher in number than the CD56 subsets and even expanded in occasional control donors but also in transporter associated with antigen processing-deficient patients, two familial hemophagocytic lymphohistiocytosis type II patients, and several common variable immunodeficiency patients. This population was detected but globally reduced in a longitudinal cohort of 18 HIV-1-infected individuals. Phenotypically, the new subset contained a high percentage of relatively immature cells, as reflected by a significantly stronger representation of NKG2A and CD57 cells compared to their CD56CD16 counterparts. The phenotype of the CD56CD16 population was differentially affected by HIV-1 infection as compared to the other NK cell subsets and only partly restored to normal by antiretroviral therapy. From the functional point of view, sorted CD56CD16 cells degranulated more than CD56CD16 cells but less than CD56CD16 NK cells. The population was also identified in various organs of immunodeficient mice with a human immune system ("humanized" mice) reconstituted from human cord blood stem cells. In conclusion, the CD56CD16 NK cell subpopulation displays distinct phenotypic and functional features. It remains to be clarified if these cells are the immediate precursors of the CD56CD16 subset or placed somewhere else in the NK cell differentiation and maturation pathway.