In rat aortic smooth muscle cells (RASMC), interferon (IFN)-gamma enhanced nitrite accumulation and type II nitric oxide synthase (iNOS) protein expression induced by interleukin (IL)-1 beta. IFN-gamma alone had no effect on nitrite accumulation or iNOS protein. IL-1 beta, but not IFN-gamma, induced nuclear factor (NF)-kappa B and CCAAT box/enhancer binding protein (C/EBP) nuclear binding. Conversely, IFN-gamma, but not IL-1 beta, induced signal transducer and activator of transcription (STAT) 1 and interferon regulatory factor (IRF)-1 binding. In a -1.4-kb rat iNOS promoter segment, deletion of an IFN-gamma-activated site (GAS) increased IL-1 beta-induced activity but inhibited IFN-gamma-enhanced activity, suggesting a two-way effect of the GAS site on iNOS induction: enhancing induction through STAT1 activation and inhibiting induction through a non-IFN-gamma-mediated mechanism. Deletion of both an IRF and a C/EBP site reduced the IL-1 beta-induced and the IFN-gamma-enhanced activities. However, IRF site mutations decreased the IFN-gamma-enhanced activity without affecting the IL-1 beta-induced activity. Insertion of two IRF sites increased the IFN-gamma-enhanced, but not the IL-1 beta-induced, activity. Mutations of a reverse NF-kappa B site did not significantly change IFN-gamma-enhanced activity. We conclude that in RASMC, NF-kappa B and C/EBP mediate the IL-1 beta-induced iNOS expression, whereas IRF-1 and STAT1 mediate the IFN-gamma-enhanced iNOS induction.