Genital reinfection with herpes simplex virus type 2 (HSV-2) is uncommon in humans. The mechanism of acquired immunity is unknown. Because the cervix is a site of HSV exposure, we measured antigen-specific T cell responses to HSV in cervical lymphocytes during both lesional and nonlesional time periods. Cells were expanded without secondary in vitro stimulation with antigen. Proliferative and cytotoxic responses to HSV were detectable in specimens from most subjects. Limiting dilution assays showed a high frequency of antigen-specific cells. Cytotoxic T cell responses included both CD4 and CD8 components. Responses were present both during and between symptomatic infection episodes and persisted during suppressive antiviral therapy. Natural infection with HSV-2 is associated with a persistent cervical mucosal cellular immune response. This local response may possibly assist in limiting the clinical consequences of secondary HSV-2 infection, whether due to endogenous reactivation or exogenous reinfection.