IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-α and nitric oxide (NO) release by using anin vitro model of intracellular infection. IL-12 alone released relatively little TNF-α and NO, whereas live mycobacteria alone released TNF-α markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-α and NO release synergistically. Because IL-12 and mycobacteria also released IFN-γ from macrophages synergistically, and exogenous IFN-γ with mycobacteria enhanced TNF-α and NO release synergistically, we examined the role of endogenous IFN-γ in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-γ, we found that IL-12/mycobacteria-enhanced macrophage TNF-α and NO release was mediated through endogenous IFN-γ. We further demonstrated that IFN-γ and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-α synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-γ release.