A study was undertaken to evaluate the efficacy of an adenoviral vector containing the murine interferon-β (IFN-β) transgene (Ad:IFN-β) against herpes simplex virus type 1 (HSV-1) infection in two transduced cell lines. The transduction of the adenoviral vector efficiency, ranging from 2% to 100%, was dependent on the multiplicity of infection (moi) (0.4-50 plaque-forming units [pfu]/cell). Supernatants from cells transduced with the Ad:IFN-β but not the adenoviral null vector (Ad:Null) contained biologically active IFN-β (6.6-106 U/ml depending on the moi). Cells transduced with the Ad:IFN-β displayed up to 25-fold reduction in viral titers compared with cells transduced with the Ad:Null or nontransduced cell controls. The suppression in viral titer correlated with a reduction in viral gene (α, β, and γ) and protein expression. The expression of IFN-β-responsive genes, including protein kinase R (PKR) and 2′, 5′-oligoadenylate synthetase (OAS), were significantly elevated in the Ad:IFN-β-transduced cells by 12-fold and 25-fold, respectively. However, after infection with HSV-1, a transient but significant drop in PKR but not OAS gene expression was observed 10 h postinfection. The absence of PKR but not RNase L significantly attenuated the antiviral efficacy of the transgene. Collectively, these results illustrate the feasibility of employing a viral vector to deliver a potent antiviral gene to targeted cells without any obvious detriment to the vector itself and support an important role for PKR as a mediator of the anti-HSV-1 activity of type I IFN.