Activation of mouse peritoneal exudate macrophages, as evidenced by destruction of the intracellular protozoan parasite Leishmania enriettii, was obtained by incubation with supernates from concanavalin A (Con A)-stimulated syngeneic spleen cells. Parasites were not destroyed in macrophages exposed to control media. Supernate-induced activation was independent of the presence of Con A. The activating principle (macrophage activating factor, or MAF) was produced by Con A-stimulated lymphocytes in presence or absence of serum. In absence of serum, MAF synthesis was highest at Con A concentrations far below those required in serum-containing media. MAF production was reduced at Con A concentrations of 10 microgram/ml or above, probably a result of toxicity of the lectin for lymphocytes. MAF was detectable after 24 h of lymphocyte stimulation and increased up to 72 h; production appeared to be independent of DNA synthesis. Serum-free MAF was inactive when tested as such on macrophages. Full activity could be restored by addition of nanogram amounts of endotoxin or of FCS before assay. Endotoxin also considerably potentiated MAF activity in serum-containing supernates. Full intracellular parasite destruction was observed after contact of macrophages with MAF for 20 h. The continuous presence of MAF was not necessary for activation; a 10-h pulse was sufficient to induce macrophages to destroy all intracellular microorganisms within the next 38 h.