Type I interferons (IFN) constitute one of the initial and most potent components of the innate immune response against viral infections. While there is only one IFN-β gene, there are several IFN-α genes whose products act through the same receptor calling into question the role of these gene products against viral infection. The focus of the present study was to compare the anti-viral state of cells transiently transfected with different murine type I IFN transgenes including IFN-α1, -α4, -α5, -α6, -α9, and IFN-β. Transfected cells produced biologically active IFN ranging from 6 to 46 units/ml. L929 and 3T12.3 cells transfected with the IFN-β transgene consistently showed a 2-4 fold reduction in herpes simplex virus type 1 (HSV-1) and HSV-2 viral titers compared with cells transfected with the IFN-α transgenes which were much less consistent based on HSV species and cell type. Parallel with the reduction in viral titers, cells transfected with the IFN-β transgene showed the complete absence or significant reduction in viral immediate early, early, and late gene expression. Collectively, the results suggest that the IFN-β transgene is superior to IFN-α transgenes against HSV infection in vitro in part due to a reduction in viral gene expression. These results indicate events downstream of the type I IFN receptor distinguish between the subtypes of IFN-α species relative to the activation of genes ultimately responsible for the establishment of the anti-HSV state.