In this study we investigate the role of the protein inhibitor of NOS-1 (PIN) in the interferon-gamma (IFN-gamma)-mediated posttranscriptional accumulation of nitric oxide synthase-1 (NOS-1) and the anti-vesicular stomatitis virus response in neuronal cells. IFN-gamma-induced enhancement of NOS-1 activity is crucial for its antiviral activity in the central nervous system. IFN-gamma treatment of neuronal cells results in an increase of total NOS-1 and decrease of total PIN proteins without alteration in their respective mRNA levels. PIN/NOS-1 complexes decreased after IFN-gamma treatment. Transfection of cells with small interfering RNA (siRNA) for PIN results in a higher constitutive activity of NOS-1 and inhibition of viral replication. IFN-gamma treatment did not change the amount of NOS-1 detectable by Western blot, when PIN is diminished by RNAi treatment. Overexpression of PIN results in lower constitutive NOS-1 expression and activity, and diminishes activation of NOS-1 by IFN-gamma. Our findings indicate that in neurons, IFN-gamma upregulates NOS-1 through proteasomal degradation of PIN.