Intravaginal inoculation of mice with an attenuated strain of herpes simplex virus type 2 (HSV-2) resulted in vigorous HSV-specific immune responses that protected against subsequent challenge with fully virulent HSV-2 strains. Even in the presence of high titers of HSV-specific Ab, T cell-dependent mechanisms were required for protection of the vaginal mucosae of HSV-immune mice and could be detected by 24 h after intravaginal reinoculation. Depletion of specific T cell subsets from HSV-immune mice before HSV-2 reinoculation demonstrated that CD4+ T cells were primarily responsible for this protection. Similarly, optimal protection of the sensory ganglia against reinfection with HSV-2 was dependent on the presence of T cells. Infectious HSV-2 was not detected in the sensory ganglia or spinal cord of HSV-immune mice depleted of only CD4+ or CD8+ T cells, suggesting that the T cell-mediated protection could be provided by either subset. Similarly, neutralization of IFN-gamma during challenge of HSV-immune mice resulted in diminished protection of the vaginal mucosa, but not of the sensory ganglia. These results suggest that the ability to induce vigorous HSV-specific T cell responses is an important consideration in the design of vaccines to protect both the vaginal mucosa and sensory ganglia against HSV-2.