Although researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) gamma and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-alpha serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-alpha also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-alpha therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-alpha treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-alpha-treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-alpha treatment of cells in vitro or administration of IFN-alpha to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-alpha in hepatitis C infection.