The presence of micromolar concentrations (5 to 100 microM) of adenosine 5'-triphosphate (ATP) in cytotoxicity assays of human natural killer (NK) cells with K562 targets resulted in marked inhibition of NK activity. NK activity of peripheral blood mononuclear cells (PBL) and of purified NK cells (i.e., large granular lymphocytes (LGL] were similarly sensitive to inhibition by ATP. The inhibitory activity was specific to ATP and was not demonstrated with GTP, UTP, CTP, or with other adenosine compounds. This inhibitory activity resulted from an effect of ATP on the effector cells and was not dependent on serum components. ATP did not inhibit binding of the LGL to target cells, and therefore the inhibition by ATP is presumed to be related to some post-recognition metabolic events. Some physiologic role in regulation of NK activity by ATP seems possible, because nonspecific phosphatases (bacterial alkaline phosphatase or human alkaline phosphatase) stimulated NK activity and partially reversed the ATP-induced inhibition of reactivity.