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Autologous mixed lymphocyte cultures were set up from nylon non-adherent T-enriched lymphocytes and mitomycin C-treated spleen cells of individual ACI/N rats and the effect on the reaction (AMLR) of sera in the culture medium was studied with regard to the xenogeneic nature of the sera. Not only foetal calf serum, but also sera of adult human, horse and swine stimulated the AMLR response, but autologous or rat serum did not. Albumin fractions of these sera were also effective in inducing the AMLR response. Furthermore, the presence of xenogeneic serum in the culture medium was also required for the blastogenic response of AMLR-primed lymphocytes against the secondary stimulation with syngeneic spleen cells. Both autologous and xenogeneic sera, however, supported the Con-A response of the same responder cell population as used in the AMLR. These results substantiated our previous finding (Endho & Hashimoto, 1981) and suggest that two signals, one from autologous non-T cells and another from the xenogeneic factor in culture medium, are required to cause AMLR, at least in the rat system. [ABSTRACT FROM AUTHOR]