Pretreatment of murine (BALB/3T3) cells with either murine or recombinant hybrid human B/D interferon (IFN) blocked the release of infectious herpes simplex virus type 1 (HSV-1) from treated cells. The block in replication was not due to an effect on attachment of HSV-1 to the target cells or to toxic effects of IFN. Immunoblot analyses showed that murine IFN significantly reduced the expression of virus-specific proteins in IFN-treated cells. In contrast, B/D IFN had no major effect on the expression of viral proteins in treated cells. In support of the above observation, electron microscopy of virus-infected cells displayed formation of nucleocapsids within the nucleus of IFN-treated cells. However, the expression of glycoproteins B and D was reduced in B/D IFN-treated cells. These results suggested that murine IFN blocked HSV-1 replication at an early stage whereas B/D IFN inhibited HSV-1 replication at a late stage in virus morphogenesis.