An antiserum, (anti-lymphokine globulin, ALyG) directed against highly purified products of activated lymphocytes, inhibits the proliferation of responding cells in guinea pig mixed leukocyte cultures (MLC). This serum recognizes three newly synthesized lymphocyte products (one of them being migration inhibition factor (MIF) which are involved in the mediation of delayed hypersensitivity reactions in vivo. Since ALyG does not appear to contain cytotoxic antibodies against guinea pig lymphocyte antigens and its inhibitory activity cannot be removed by absorption with lymphoid cells it seems likely that the inhibition of MLC reactivity is not mediated by the lysis of stimulator or responder cells and its target is not the lymphocyte per se but possibly some factor elaborated during MLC response. By contrast, antisera with specificity for histocompatibility (H) antigens can inhibit the MLC when the appropriate H antigens are present on the responding and/or stimulating cell population. However, this inhibitory activity of the antisera can be effectively absorbed with lymphoid cells bearing the appropriate H antigens. The addition of ALyG even 48 hr after the initiation of culture results in a marked inhibition of MLC reactivity. This finding is consistent with the elaboration of a mitogenic factor or signal during the first 48 hr of culture and the delivery of this signal to the responding cell population. Thus, ALyG does not appear to interfere with the synthesis of this factor but must be present after its release in order to block responder cell proliferation. Furthermore, the stimulatory effect of this MLC mitogenic factor, but not of PPD-induced mitogenic factor on “third party” cells can be completely inhibited when the cells are cultured in the presence of ALyG. These findings suggest that the MLC-mitogenic factor in this system is identical to or cross-reactive with one or both of the newly synthesized molecules recognized by the ALyG.