Interferon (IFN) is an important immune system molecule capable of inducing an antiviral state within cells. Herpes simplex virus type 1 (HSV-I) replication is somewhat reduced in tissue culture in the presence of IFN, presumably due to decreased viral transcription. Here,we show mutations that inactivate immediate-early (IE) gene product ICP0 render HSV-1 exquisitely sensitive to IFN inhibition, resulting in greatly decreased levels of viral mRNA transcripts and the resulting polypeptides and a severe reduction in plaque formation ability. Mutations in other HSV-1 genes, including the genes coding for virion transactivator VP16 and the virion host shutoff protein vhs, LE: gene ICP22, and the protein kinase UL13 gene, do not increase the IPN sensitivity of HSV-1. interestingly, ICP0 mutants demonstrate the same level of sensitivity to IFN as wild-type virus on U2OS cells, an osteosarcoma cell line that is known to complement mutations in ICP0 and VP16. Thus, in some cell types, functional ICP0 is required for HSV-I to efficiently bypass the inhibitory effects of IFN in order to ensure its replication. The significance of this link between ICP0 and IFN resistance is discussed.