Local mucosal cellular immunity is critical in providing protection from HSV-2. To characterize and quantify HSV-2-reactive mucosal T cells, lymphocytes were isolated from endocervical cytobrush and biopsy specimens from 17 HSV-2-infected women and examined ex vivo for the expression of markers associated with maturation and tissue residency and for functional T-cell responses to HSV-2. Compared with their circulating counterparts, cervix-derived CD4+ and CD8+ T cells were predominantly effector memory T cells (CCR7-/CD45RA-) and the majority expressed CD69, a marker of tissue residency. Co-expression of CD103, another marker of tissue residency, was highest on cervix-derived CD8+ T cells. Functional HSV-2 reactive CD4+ and CD8+ T-cell responses were detected in cervical samples and a median of 17% co-expressed CD103. HSV-2-reactive CD4+ T cells co-expressed IL-2 and were significantly enriched in the cervix compared with blood. This first direct ex vivo documentation of local enrichment of HSV-2-reactive T cells in the human female genital mucosa is consistent with the presence of antigen-specific tissue-resident memory T cells. Ex vivo analysis of these T cells may uncover tissue-specific mechanisms of local control of HSV-2 to assist the development of vaccine strategies that target protective T cells to sites of HSV-2 infection.