Natural killer (NK) cells are physiologically involved in the immune response against viruses, intracellular bacteria, and parasites as well as against malignant diseases. In addition to the cytotoxic activity, NK lymphocytes mediate a variety of homeostatic effects by producing cytokines. This study focused on the differential role of CD40 and CD80 costimulatory molecules and major histocompatibility complex class I (MHC‐I) antigens in the regulation of cytotoxicity and of interferon (IFN)‐γ secretion of resting and interleukin (IL)‐2‐activated human NK cells. CD40 and CD80 molecules were observed to play a specific role in the induction of cytotoxic function but not in IFN‐γ production of IL‐2‐activated NK effectors. In addition, a critical role of CD94‐dependent MHC‐I recognition for the regulation of IFN‐γ production and target lysis was demonstrated. These data provide a possible mechanism underlying functional interactions between NK lymphocytes and CD40/CD80‐expressing cell targets, as represented by dendritic cells.