Previously, we reported that exposure of bone marrow-derived macrophages (MΦ) to a phagocytic stimulus in the simultaneous presence of interferon-γ (IFN-γ) induced these cells to generate nitrite (NO2). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettü) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2 secretion. As shown here, the capacity of phagocytosis to elicit NO2 production by IFN-γ-treated MΨ was inhibited by antibody to murine recombinant tumor necrosis factor-α (rTNF-α), suggesting that phagocytosis enabled IFN-γ to activate MΨ via the induction of TNF-α as an autocrine second signal, Mϕ NO2 production in response to rIFN-γ and either exogenous TNF-α or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism, However, addition of either Leishmania promastigotes or latex beads to Mϕ cultures simultaneously exposed to both IFN-γ and exogenous murine or human rTNF-α further potentiated activation as measured by NO2 release. Furthermore, anti-TNF antibody failed to inhibit Mϕ responses to rIFN-γ and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-α did not significantly affect NO2 production by IFN-γ/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances Mϕ responses by a process more complex than the sole induction of TNF-α. Phagocytosis also increased Mϕ NO2 production elicted by IFN-γ plus TNF-α in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating Mϕ microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.