Activated mouse macrophages can produce high levels of nitric oxide, an antimicrobial effector molecule recently also implicated in antiviral defense. As viral infection may alter macrophage functions, nitric oxide production was investigated in murine macrophages infected with a Flavivirus, tick-borne encephalitis virus. Infected macrophages produced high levels of nitric oxide upon stimulation with lipopolysaccharide without priming, while in control macrophages induction of nitric oxide production by lipopolysaccharide required priming with interferon-gamma. Addition of interferon-gamma to infected macrophages further increased lipopolysaccharide-stimulated nitric oxide production. In contrast, nitric oxide production upon stimulation with interferon-gamma plus tumor necrosis factor-alpha was markedly reduced in infected macrophages. Downregulation of interferon-gamma plus tumor necrosis factor-alpha-induced nitric oxide synthesis by viral infection could be attributed to endogenous interferon-alpha beta produced by infected macrophages. Addition of interferon-alpha beta to uninfected macrophages inhibited interferon-gamma plus tumor necrosis factor-alpha-induced nitric oxide production, and addition of interferon-alpha beta antibodies to infected macrophages increased identically stimulated nitric oxide production to normal levels. Thus, interferon-alpha beta mimicked the effect of viral infection on macrophage nitric oxide production. These findings indicate that viral infection profoundly alters requirements of mouse macrophages for induction of nitric oxide synthesis, depending on the activating signal applied. The described effects of viral infection of macrophages on the regulation of nitric oxide production and new complexity to the role of nitric oxide in host defense against viruses.