Challenge of elicited peritoneal macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was followed by an apoptotic response. These cells expressed cytokine-inducible nitric oxide synthase (iNOS) in response to these stimuli, and the NO released contributed markedly to the apoptotic death, as deduced from the increased viability observed when iNOS activity was inhibited. The antiviral type I IFN (IFN-α/β) downregulated the high levels of NO produced when cells were stimulated with suboptimal doses of LPS and IFN-γ. Moreover, IFN-α/β also decreased cell death in LPS/IFN-γ-activated cells, as determined by the reduction in the content of oligonucleosomal DNA fragments, in the binding of annexin V to the plasma membrane, and in the amount of hypodiploid cells when analyzed by flow cytometry after in vivo staining with propidium iodide. Kinetic analysis of the protection exerted by IFN-α/β against the apoptosis induced by treatment with LPS and IFN-γ showed that type I IFNs were very effective when added up to 1 h after IFN-γ/LPS stimulation. Addition of IFN-α/β 4 h after stimulation with IFN-γ/LPS failed completely to prevent apoptosis. This inhibition of apoptosis elicited by IFN-α/β suggests the existence of a mechanism intended to improve macrophage viability in the course of certain viral infections.