To evaluate the effects of the route of vaccination on protection against genital HSV infection guinea pigs were vaccinated twice with vaccinia recombinants expressing HSV-2 glycoprotein D or controls by intradermal, intranasal or intravaginal inoculation.. Following HSV-2 intravaginal challenge, immunization by all 3 routes reduced vaginal HSV shedding and acute genital disease. The intravaginal vaccination, however, most effectively reduced primary and recurrent HSV disease. Vaccinia and HSV antibody titers were similar in all vaccinia gD immunized groups while the lymphoproliferative response was highest in the intradermal group. In a smaller follow-up experiment we demonstrated that intrarectal immunization with vaccinia gD could also induce antibody and T cell responses.