OM-89, a proteinaceous extract from Escherichia coli with very low endotoxin content, was tested for its capacity to stimulate in vitro cells involved in the immune response. OM-89 induced a marked proliferation of mouse spleen cells; E. coli lipopolysaccharide (LPS) at the same concentration as present in OM-89 was totally ineffective. Passage through nylon wool strongly decreased the OM-89-induced effect, suggesting that the responding lymphocytes were of the B lineage. Exposure of bone marrow-derived macrophages to OM-89 promoted glucose oxidation through the hexose monophosphate shunt pathway and the capacity to generate superoxide upon phorbol myristate acetate (PMA) stimulation. These effects were not blocked by polymyxin B, whereas this compound completely prevented induction of similar metabolic activation by E. coli lipopolysaccharide. In addition, OM-89 treatment induced marked PMA-dependent superoxide and hydrogen peroxide release by macrophages from the LPS low responder mouse strain C3H/HeJ. Incubation with recombinant murine interferon-gamma and OM-89, but not with either compound alone, led to functional activation, as shown by the killing of tumor target cells, and by the destruction of the intracellular parasite Leishmania enrietti by macrophages of both LPS-responsive and unresponsive mouse strains. These experiments indicate that OM-89 can stimulate metabolic and functional activities of lymphocytes and macrophages that are important for host defense.