Spleen cells of untreated mice became nonspecifically cytotoxic after cultivation in the presence of a streptococcal preparation, OK-432, for 3 or more days. The cytotoxic cells, named OK-432-induced killer (OIK) cells, injured a variety of target cells including syngeneic EL-4 cells resistant to natural killer (NK) cells, but not NK-susceptible YAC-1 cells. C57BL/6 splenic cells cultured for 5 days in the presence of both OK-432 and interleukin 2 (IL-2) were highly cytotoxic to both EL-4 and YAC-1 cells, and had Thy-1+, Lyt-1,2-, and asialogGM1+ phenotypes, which were identical with those of OIK cells. A test for competitive inhibition with cold target cells and fractionation by centrifugation onto discontinuous density gradients of Percoll showed that cytotoxic cells generated by OK-432 plus IL-2 comprised at least two populations; the cells in the first population were cytotoxic to EL-4 cells but not to YAC-1 cells, and were smaller in size than those in the second population, which were cytotoxic to YAC-1 cells and had NK morphology. Therefore, generation of OIK cells was augmented by IL-2.