Immunization of mice with herpes simplex virus type 1 (HSV-1) mutant viruses containing deletions in the gene for virion host shutoff (vhs) protein diminishes primary and recurrent corneal infection with wild-type HSV-1. vhs mutant viruses are severely attenuated in vivo but establish latent infections in sensory neurons. A safer HSV-1 mutant vaccine strain, Delta41Delta29, has combined vhs and replication (ICP8-) deficits and protects BALB/c mice against primary corneal infection equivalent to a vhs- strain (BGS41). Here, we tested the hypothesis that Delta41Delta29 can protect as well as BGS41 in a therapeutic setting. Because immune response induction varies with the mouse and virus strains studied, we first determined the effect of prophylactic Delta41Delta29 vaccination on primary ocular infection of NIH inbred mice with HSV-1 McKrae, a model system used to evaluate therapeutic vaccines. In a dose-dependent fashion, prophylactic Delta41Delta29 vaccination decreased postchallenge tear film virus titers and ocular disease incidence and severity while eliciting high levels of HSV-specific antibodies. Adoptive transfer studies demonstrated a dominant role for immune serum and a lesser role for immune cells in mediating prophylactic protection. Therapeutically, vaccination with Delta41Delta29 effectively reduced the incidence of UV-B-induced recurrent virus shedding in latently infected mice. Therapeutic Delta41Delta29 and BGS41 vaccination decreased corneal opacity and delayed-type hypersensitivity responses while elevating antibody titers, compared to controls. These data indicate that replication is not a prerequisite for generation of therapeutic immunity by live HSV mutant virus vaccines and raise the possibility that genetically tailored replication-defective viruses may make effective and safe therapeutic vaccines.