Macrophages infected with Brucella abortus are able to kill intracellular brucellae over the first 12 to 24 hr following infection. Thereafter, the surviving brucellae replicate. We have shown previously that macrophages activated with interferon-gamma (IFN-gamma) have enhanced brucellacidal and brucellastatic activities. In studies reported here, treatment of macrophages with methylene blue, an electron carrier, enhanced the initial killing of intracellular brucellae, indicating their susceptibility to reactive oxygen intermediates. In addition, inhibitors of reactive oxygen intermediates partially blocked the antibrucella activities exhibited by both non-cytokine-treated and IFN-gamma-activated macrophages. In contrast, addition of up to 5 mM NG-monomethyl-L-arginine, to block generation of nitric oxide, resulted in minor but significant levels of blocking of macrophage antibrucella activities only when macrophages were not maximally activated even though maximally activated macrophages produced nitric oxide as indicated by accumulation of nitrite in culture supernatants. In addition, while the J774A.1 macrophage cell line had antibrucella activities which were enhanced by IFN-gamma activation, it did not produce nitric oxide when activated with IFN-gamma and infected with B. abortus. Finally, the IFN-gamma-induced enhancement of antibrucella activities by peritoneal macrophages was inhibited by addition of antitumor necrosis factor-alpha (TNF-alpha) antibodies to the cultures, indicating that TNF-alpha is necessary for full expression of the macrophage antibrucella activities.