We examined the contributions of reactive nitrogen and oxygen intermediates (RNI and ROI, respectively) in macrophage bactericidal activity against Burkholderia pseudomallei, the causative agent of melioidosis, in order to understand host defense mechanisms against infection caused by this bacterium. The bacteria multiplied in unstimulated murine macrophage cell line J774.1. However, a strong dose-dependent inhibition of intracellular bacterial growth was observed when gamma interferon (IFN-gamma)-activated macrophages were used. The induction of bactericidal activity correlated well with the production of nitric oxide (NO) by IFN-gamma-activated macrophages and was markedly suppressed by N(G)-monomethyl L-arginine (L-NMMA), a competitive inhibitor of NO synthesis. Superoxide dismutase (SOD) and catalase significantly inhibited macrophage bactericidal activity, and the combined addition of L-NMMA, SOD, and catalase resulted in the complete inhibition of IFN-gamma-stimulated activity. The bacteria were susceptible to the killing effects of chemically generated NO and superoxide anion in a macrophage-free system. Our results indicate that IFN-gamma-induced macrophage bactericidal activity against B. pseudomallei is mediated to a large extent by RNI killing mechanisms and to a lesser extent by ROI-dependent mechanisms.