Platelet activating factor (PAF) was found to suppress primary and secondary mixed lymphocyte reactions and BN52021, a naturally occurring PAF antagonist, blocked PAF-mediated suppression and enhanced the mixed lymphocyte reactions. The effect of delayed addition of PAF or BN52021 24 h or later after the initiation of cultures reduced the suppressive and enhancing effects, respectively. The removal of the antagonist BN52021 from mixed lymphocyte cultures up to 72 h after their initiation also was found to eliminate the potentiating effect of this antagonist. The continuous presence of PAF in mixed lymphocyte cultures used to generate cytotoxic lymphocytes suppressed the generation of effector cells while the addition of BN52021 elicited an enhanced level of cell-mediated cytotoxicity in such cultures. BN52021 enhanced the cytotoxic activity in such cultures irrespective of the presence of exogenous interleukin-2, suggesting that the antagonist-mediated enhancement is not due to the enhanced production of interleukin-2 by cells in the mixed cultures. The experiments reported here provide evidence for a role of PAF in modulating the complex interactions that take place in the initiation of cellular immune reactions. Furthermore, the results of these experiments indicate that the immunoregulatory action of PAF can be modulated by an antagonist that exerts its action independently of the production of the lymphocyte growth factor, interleukin-2.