Abstract An enriched population of human natural killer (NK) cells was obtained by density gradient centrifugation. The cytotoxic activity of these cells was enhanced by pretreatment with human leukocyte interferon (IF), and the metabolic requirements of this enhancement were examined. Augmentation of NK activity was initiated in a rapid, temperature-independent manner, requiring only a 10- to 15-min exposure to IF, and occurred at either 4 or 37°C. Increased activity of the IF-treated NK effector cells was consistently observed after only 30 min of contact with target cells. Augmentation was inhibited by prior treatment of NK effector cells with actinomycin D (AD), but treatment with AD after 1 hr of IF treatment did not inhibit the IF-mediated increase in cytotoxicity, suggesting that the RNA species required for enhancement are synthesized within 1 hr of cell-IF interaction. Protein synthesis was required for at least 1 hr following cell-IF interaction, as shown by the ability of emetine and puromycin treatments to abrogate increased NK cell activity. Binding of IF to cells was independent of protein synthesis. IF-induced enhancement was unaffected by incubation of effector cells with mitomycin C either before or after IF treatment, indicating that IF acts primarily upon a population of preexisting cells of lower NK activity.