Murine thymus-derived (T) cells may be divided into two distinct subgroups on the basis of differences in their relative density of the T differentiation antigen theta (ϑ), differential responsiveness to the T dependent mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) as well as by differences in recirculation patterns and radiation sensitivities. Thus, among splenic lymphocytes one population of T cells bears a relatively high density of ϑ determinants, demonstrates relatively equal responsiveness to PHA and to Con A, resides within the recirculating pool of lymphocytes and is relatively radiation sensitive. Another population of splenic T cells bears a relatively reduced density of ϑ determinants, demonstrates reactivity predominantly to Con A and belongs to a sessile, radiation resistant lymphocyte pool. The ability to delineate T cell subgroups by these characteristics provides a useful means of investigating the functional characteristics of T cell subsets. In this report, we demonstrate that T cells from the spleens of nonimmunized mice which are reactive in one-way allogeneic mixed lymphocyte cultures (MLC) belong to a T cell subset distinct from that population of T cells, present in spleens of immune mice, which is capable of lysing allogeneic cells. Thus, the administration of limiting amounts of anti-thymocyte serum (ATS) or sublethal irradiation to normal C57BL/6 mice removes the recirculating, radiation sensitive population of cells. The residual spleen T cell population does not manifest mixed lymphocyte reactivity to (C57BL/6 × BALB/c)F1 spleen cells. On the other hand, the cytolytic activity of spleens from alloimmunized C57BL/6 mice is unaffected by these treatments. Irradiation or ATS treatment before immunization, however, completely ablates the development of cytolytically active splenic lymphocytes. Thus, although the MLC reactive cells and the cytolytically active cells belong to different splenic T cell populations, the MLC cell appears to be required to generate cytolytic activity. Possible mechanisms to explain this requirement are presented.