The mechanisms involved in host clearance of symptomatic mucocutaneous herpes simplex virus (HSV) infection are unclear. We studied the functional properties of bulk cultures of skin-infiltrating lymphocytes from normal skin and serial biopsies of recurrent genital HSV-2 lesions, and compared HSV-specific and NK responses with viral clearance. HSV-specific CD4+ or CD8+ T cells were rarely detected in lymphocytes cultured from normal skin. The total lymphocyte count and HSV-specific and NK-like effector cell activities were markedly higher in cultures derived from lesional skin. HSV-specific CD4+ proliferative responses and NK-like cytotoxic responses were present at all stages of herpetic lesions, including biopsies early in the disease course. In contrast, cytotoxic T lymphocyte activity was generally low among cells derived from early culture-positive lesions, and increased during lesion evolution. Viral clearance from the lesion site was associated with a high level of local cytolytic activity towards HSV-infected cells. The phenotypes of cells with HSV-specific cytotoxic responses varied between patients, having CD4+ and CD8+ components. Immunotherapeutic approaches to HSV should be directed at improving in vivo cytolytic activity to HSV.