This investigation examined the effects of mediators derived form activated spleen cells on macrophage Ia-antigen expression and function. Incubation of adherent thioglycollate-induced murine peritoneal macrophages(> 90% Ia-) with concanavalin A (Con A)-stimulated spleen cell supernate (Con A sup) resulted in a dose-dependent increase in the percentage of Ia-containing (Ia+) phagocytic cells, as detected by antiserum-and-complement-mediated cytotoxicity. The Ia-antigen expression of macrophages incubated with unstimulated spleen cell supernate supplemented with Con A (Control sup) declined. Pretreatment of the macrophages with anti-Ia and complement before addition of the Con A sup did not inhibit subsequent Ia-antigen expression, suggesting that Ia- macropohages were converted to Ia+ cells. These findings were not a result of adsorption of soluble Ia-antigen from the Con A sup, because Ia-antigen expression was detected by an antiserum specific for the haplotype of the macrophages but not that of the allogeneic spleen cells from which the supernate was prepared. Con A sup-cultured macrophages also stimulated the proliferation of allogeneic spleen cells significantly better than Control sup-cultured macrophages in the mixed leukocyte reaction (MLR). Pretreatment of Con A sup-cultured macrophages with anti-Ia and complement before addition of splenic responder cells abrogated their stimulatory capacity, indicating the Ia dependence of the MLR. We hypothesize that regulatory lymphokine(s) can induce both the expression of the Ia+ phenotype by macrophages and the functional capability to stimulate the MLR, and that macrophages lose these capabilities in the absence of such mediator(s).