The mechanism of alloantigen activated spleen cell suppression of mixed lymphocyte reaction (MLR) was explored. Activated murine suppressor spleen cells elaborated a soluble noncytotoxic factor which suppressed MLR responses by 55-95%. Generation of suppressor factor required both in vivo alloantigen sensitization and specific in vitro restimulation. Suppressor factor was not produced by activated spleen cells which had been treated with anti Thy 1.2 serum and complement. Antigenic specificity toward alloantigens of the stimulator cells was not demonstrable. In contrast, suppressor factor effectively inhibited MLR responses only of responder cells of those strains that shared the D end and the I C subregion of the H 2 complex with the cells producing suppressor factor. Therefore, active suppression appears to require an MHC directed homology relationship between regulating and responder cells in MLR.