The influence of Ca2+ on the cytotoxic activity of natural killer cell cytotoxic factor (NKCF) was analyzed. When the natural killer susceptible cell line K562 was exposed to NKCF in the presence of 5 mM Ca2+, two peaks of cell damage were found. The first peak was observed after 30-40 min of incubation as a result of pore formation on the surface of target cells. The second was a peak of cytolytic activity which appeared after 24 h of incubation. Upon dilution of the NKCF preparation, only the first peak was observed. Therefore, NKCF produced by large granular lymphocytes in response to K562 consists of different proteins and represents pore-forming and cytolytic activities.