A subset of CD3(neg)CD56(neg)CD16 + Natural Killer (NK) cells is highly expanded during chronic HIV-1 infection. The role of this subset in HIV-1 pathogenesis remains unclear. The lack of NK cell lineage-specific markers has complicated the study of minor NK cell subpopulations. Using CD7 as an additional NK cell marker, we found that CD3(neg)CD56(neg)CD16 + cells are a heterogeneous population comprised of CD7 + NK cells and CD7(neg) non-classical myeloid cells. CD7 + CD56(neg)CD16 + NK cells are significantly expanded in HIV-1 infection. CD7 + CD56(neg)CD16 + NK cells are mature and express KIRs, the C-type lectin-like receptors NKG2A and NKG2C, and natural cytotoxicity receptors similar to CD7 + CD56 + CD16 + NK cells. CD7 + CD56(neg) NK cells in healthy donors produced minimal IFNγ following K562 target cell or IL-12 plus IL-18 stimulation; however, they degranulated in response to K562 stimulation similar to CD7 + CD56 + NK cells. HIV-1 infection resulted in reduced IFNγ secretion following K562 or cytokine stimulation by both NK cell subsets compared to healthy donors. Decreased granzyme B and perforin expression and increased expression of CD107a in the absence of stimulation, particularly in HIV-1-infected subjects, suggest that CD7 + CD56(neg)CD16 + NK cells may have recently engaged target cells. Furthermore, CD7 + CD56(neg)CD16 + NK cells have significantly increased expression of CD95, a marker of NK cell activation. Taken together, CD7 + CD56(neg)CD16 + NK cells are activated, mature NK cells that may have recently engaged target cells.