Normal mice contain cytolytic cells with specificity for in vitro grown mouse Moloney leukemia cells. Such killer cells are most frequent in the spleens; lymph node and bone marrow contain less and thymus virtually no killer activity. Peak activity is found around one to three months of age. Spleen cells from genetically athymic mice are as active killer cells as those from normal mice of the same strain. Treatment with anti-theta serum plus complement followed by removal of adherent and surface Ig positive cells by filtration through anti-Ig columns will leave between 1-5% of the original spleen cell population from a normal mouse. These cells have the morphology of small lymphocytes and perhaps contain all of the total original killer activity of the spleen against the Moloney leukemia cells. Such killer enriched cells are devoid of T and B lymphocytes and largely fail to function in antibody induced, cell-mediated lysis against antibody-coated chicken erythrocytes. It is concluded that the spontaneous selective cytotoxic activity of normal mouse spleen cells against Moloney leukemia cells is exerted by small lymphocytes of yet undefined nature.