BACKGROUND: The role of mucosal immunity in defense of the female genital tract against pathogens such as herpes simplex virus-2 (HSV-2) is poorly understood. Here we explored the use of a new mouse model to determine whether local immune events in the vagina of immune animals may protect them against genital herpes. EXPERIMENTAL DESIGN: The effect of the estrous cycle, pregnancy, and sex hormones on vaginal infection of adult mice by HSV-2 was determined by immunolabeling of virus proteins. The immune response to infection was studied by immunolabeling of T lymphocytes, B lymphocytes, and plasma cells in the vagina of infected mice. RESULTS: Inoculation of attenuated virus (TK-HSV-2) or wild-type virus (TK+HSV-2) into the vagina on day 6 of pregnancy or after treatment with Depo-Provera (DP) caused infection of the vaginal epithelium. In contrast, these viruses did not cause infection after vaginal inoculation at estrus, metestrus, or after treatment with Depo-Estradiol. Infected mice showed immunolabeling of virus in the vaginal epithelium from 24 hrs to 5 days after virus inoculation. The immune response to infection included upregulation of class II MHC antigen in vaginal epithelium, CD8+ T cells in epithelium and stroma, and plasma cells and lymphoid nodules in the stroma. Mice that were infected with TK-HSV-2 did not exhibit infection of vaginal epithelium when challenged 6 weeks later with TK+HSV-2. CONCLUSIONS: Progesterone-dominated adult mice become infected after intravaginal inoculation with HSV-2, but estradiol-dominated mice are refractory. Vaginal infection with attenuated HSV-2 produces immunity that protects mice against later infection by wild-type virus. This immunity either prevents infection of vaginal epithelium or severely inhibits viral replication in the epithelium. The observations suggest that the E/DP-treated adult mouse should be a useful model for studies of mucosal immunity to vaginal infection by HSV-2.