We investigated the role of the constitutive nitric oxide (NO) in the expression of interferon (IFN) genes in mouse peritoneal macrophages (PM). The treatment of PM with l-arginine-NG-amine (AA), a potent inhibitor of NO-producing enzymes, resulted in a marked accumulation of IFN-α4 mRNA and, to a minor extent, of IFN-β mRNA. In contrast, the expression of IFN-γ mRNA, as well as tumor necrosis factor alpha and interleukin-6 mRNA, was not affected. Furthermore, a remarkable increase in the expression of the IFN regulating factor 1 (IRF-1), but not of IRF-2, mRNA was detected in AA-treated PM. To investigate whether the AA-induced activation of the IFN system correlates with the production and antiviral activity of IFN, the extent of encephalomyocarditis virus (EMCV) replication was monitored in AA-treated PM with respect to control cultures. AA treatment strongly inhibited, in a dose-dependent manner, EMCV yields in PM. Likewise, similar results were obtained by the addition of the NO-scavenger carboxyphenyl-tetramethylimidazoline-oxyl-oxide. In addition, inhibition of NO synthesis by NG-mono-methyl-l-arginine in PM strongly decreased virus replication in coculture of PM and EMCV-infected L929 cells, whereas no antiviral effect was observed in L929 cells alone. Moreover, the AA-mediated antiviral activity was abrogated in the presence of antibody to IFN-α/β, whereas antibody to IFN-γ was completely ineffective. Taken together, these results indicate that low levels of NO, constitutively released by resting PM, negatively regulate the expression and activity of IFN-α/β in PM. We suggest that NO acts as a homeostatic agent in the regulation of IFN pathway expression in macrophages.