Abstract We have examined the effects of lipopolysaccharide (LPS) and the double stranded RNA, polyadenylic:polyuridylic acid (poly A:U) on the generation of cytotoxic T lymphocytes in a mixed leukocyte culture (MLC) reaction of allogeneic mouse spleen cells. Both LPS and poly A:U augment the generation of cytotoxic cells in responder-stimulator combinations, which differ for the entire H-2 region or only for the H-2K or H-2D subregion. Also, in the presence of optimal concentrations of LPS and poly A:U the dose of antigen required for optimal sensitization and generation of cytotoxic cells is reduced significantly. The cytotoxic cells generated are specific for the sensitizing antigen and they resemble the cytotoxic T lymphocytes in a normal MLC in that they show a log-linear relationship between effector:target ratio and percent cytotoxicity. Furthermore, both LPS and poly A:U appear to bring about their effect by acting on the responding cells as the enhanced generation of cytotoxic cells is brought about if the responding cells are preincubated with these agents and washed before adding the stimulating cells. The effect of poly A:U depends on the presence of adherent cells whereas the effect of LPS is independent of the presence of these cells suggesting that the two agents exert their effect at different steps in the development of the cytotoxic cells. These results suggest that elucidation of the mode of action of LPS and poly A:U in this system might shed light on the mechanisms underlying development of cytotoxic T lymphocytes in vitro.