Vaccinia virus recombinants expressing antigens from herpes simplex virus (HSV) have been tested as potential live virus vaccines for prevention of HSV infection. We describe three vaccinia virus/HSV recombinants. The first expresses the HSV-1 glycoprotein D (vaccinia/gD), the second expresses the HSV-1 glycoprotein B (vaccinia/gB), and the third expresses both the HSV-1 glycoprotein D and the influenza A hemagglutinin (vaccinia/HSVgD/influenza). Mice immunized with vaccinia/gD or vaccinia/gB developed antibodies capable of neutralizing HSV in vitro and were protected against both lethal and latent infection with HSV. Protection against HSV challenge persisted for greater than 1 year in mice immunized with vaccinia/gD. The immune response to HSV in mice immunized with vaccinia/gD could be increased by a booster vaccination with vaccinia/gD. However, the immune response to HSV was decreased in animals immunized with a vaccinia recombinant that expressed non-HSV genes before vaccination with vaccinia/gD. In separate experiments, a bivalent vaccinia recombinant, vaccinia/HSVgD/influenza, was constructed and was found to be comparable to the vaccinia/gD single recombinant in immunogenicity and protective efficacy against lethal HSV challenge. We conclude that vaccinia/HSV recombinants can provide protection against HSV infection in mice and that these recombinants may provide an alternative approach in the development of a live virus vaccine against HSV.