Resident mouse peritoneal macrophages were incubated in Sephadex G-100 fractions of supernates from concanavalin A-stimulated lymphocytes. A significant effect of the lymphocyte supernatant fractions containing mediators on macrophage 5'-nucleotidase, glucose-1 14C oxidation, cell maintenance, and migration is reported. The 5'-nucleotidase was depressed to an extent similar to that seen in activated macrophages obtained from Listeria-infected mice. On the other hand, glucose-1-14C oxidation was enhanced, but not to the same degree as seen in the counterparts in vivo. Whereas migration inhibitory factor (MIF) and cell adherence-augmenting activity were found in a number of adjacent fractions, the metabolic effects were found predominantly in a single fraction. Resident peritoneal macrophages or those elicited by the injection of a lymphocyte-derived chemotactic factor were more responsive with respect to the biochemical changes than caseinate-elicited macrophages. On the other hand, caseinate-elicited macrophages appeared to be more sensitive with respect to the effects of mediator(s) on cell retention. A possible dissociation between MIF and cell-adherence augmenting activity, on the one hand, and the entities that stimulate glucose-1-14C oxidation is reported, based on fractionation studies, and loss of the latter activity upon storage of lymphocyte supernates.