A murine model of genital infection with a thymidine kinase-deficient (tk-) strain of herpes simplex virus type 2 (HSV-2) was utilized to examine the development of the local T cell response in the genital mucosa and draining genital lymph nodes (gLN). HSV-specific cytokine-secreting T cells were detected in the gLN 4 days postintravaginal inoculation but not in the urogenital tract or spleen until 5 days postinoculation, suggesting the cellular immune response originates in the gLN. More CD4+ than CD8+ gLN T cells were detected by flow cytometric analysis following primary vaginal inoculation and the majority of HSV-specific gLN T cells detected by ELISPOT were CD4+ and Th1-like based on secretion of IFN gamma and not IL-4 or IL-5. A similar population of HSV-specific memory T cells persisted in the genital tract 2 months following HSV-2 tk- genital inoculation. These data suggest that the urogenital cellular immune response elicited in mice following genital inoculation with HSV-2 tk- is predominantly CD4+ and Th1-like, resembling that observed in humans. The results of this study are important for the rational design of vaccines capable of inducing protective immunity in the genital tract.