Spleen cells obtained from BALB/c mice 4 days after allosensitization to C57BL/6 spleen cells via footpad injection suppressed the in vitro generation of BALB/c cytotoxic lymphocytes (CL) against C57BL/6 spleen cells in mixed leukocyte cultures (MLC). Suppressor activity was demonstrated by spleen cells at 4 and 7 days but not at 2, 10, or 14 days after allosensitization and was abolished by treatment with anti-Thy-1.2 serum and complement. A weak and transient cytotoxic response directed against the sensitizing alloantigen was associated with suppressor spleen cell populations, but was dissociated from suppressor function by two experimental approaches. First, increasing stimulatory cell concentration in MLC did not competitively diminish the suppressor activity; rather, the magnitude of suppression increased as the stimulatory cell concentration was increased. Second, BALB/c suppressor cells generated in vivo by either H-2(b) or H-2(k) alloantigens suppressed CL responses generated simultaneously against both alloantigens in vitro. CL responses generated against one or the other H-2 haplotype in vitro were suppressed only by suppressor cells activated by that haplotype. Therefore, splenic suppressor cells activated by alloantigen in vivo required antigen-specific restimulation in vitro; thereafter, responder cells syngeneic with the suppressor cell were rendered hyporesponsive to alloantigens by an antigen-nonspecific mechanism.