The effect of interferon (IFN) treatment on the early stages of herpes simplex virus type 1 (HSV-1) replication in three types of human cells was investigated. Interferon pretreatment was shown to reduce the steady state levels of both total and polysomebound HSV-1 immediate early α mRNAs. Using the nuclear run-off transcription assay, we showed that IFN selectively inhibited transcription of the HSV-1 genes, with no effect on transcription of total cellular RNA or that of the β-tubulin RNA. Thus, IFN appears to inhibit the initiation of HSV-1 α gene transcription rather than affect the stability of the respective mRNAs. IFN did not prevent the HSV-1 induced early shut-off of host cellular protein synthesis caused by a structural protein of the infecting virus. This observation indicated that the IFN-mediated inhibition of HSV-1 replication is at a stage beyond viral penetration into the cytoplasm. These results suggested that IFN blocked HSV-1 replication primarily at a very early stage, during the onset of α mRNA transcription.