The activity of NK cells in 12 healthy male donors was studied after 4, 8, 12, 16 and 20 h incubation of killer (mononuclear) cells with K562 target cells; a modification of the standard 3-5 h NK assay was used without preliminary centrifugation of the plates. The results indicated that 4 or 8 h incubation of killer and target cells (ratio 50:1) did not achieve maximum activity of NK cells. A significant increase in activity was found up to 12 h incubation. It was noted that variation in values decreased as the incubation time increased. The optimal incubation period was 16 h at which time maximum 51Cr release and least variation were registered. Longer incubation of K562 cells also contributed to their higher sensitivity to NK cells. The experimental data revealed in vitro factors responsible for fluctuations in the level of lytic NK activity in repeated assays in the same healthy donors.