Spleen cells from untreated young male and female C57BL/6 and C58 mice and of male C3H/He mice showed cytotoxic activity in vitro against BALB/c X-radiation-induced leukemia RL male 1 cells by 51Cr-releasing lymphocyte-mediated cytoxicity (LMC) tests, but old mice of these strains lacked LMC activity. In contrast, spleen cells from male and female AKR, BALB/c, and DBA/2 mice, and from female C3H/He mice had no appreciable LMC activity. The proportion of active cells in spleens from young (C57BL/6 times BALB/c)F1 or reciprocal hybrid mice was higher in females than in males. The specificity of the LMC reaction of RL male 1 cells, determined by LMC inhibition assays, was somewhat different from that of previously reported serologic X.1 tests. Thus the antigen detected by LMC has been tentatively designated X.1'. The main effector cells in this system were uncharacterized cells not adherent to glass surfaces or nylon-wool columns. These findings in RL male 1 leukemia extend the evidence for the presence of naturally occurring LMC. With the single unexplained exception of strain C3H/He, the LMC activity against RL male 1 cells, exhibited by untreated mice of various strains, corresponded with a previous classification of mouse strains immunologically as X.1 responders or as X.1 nonresponders according to their ability to reject X.1-positive leukemia cells.