In the present study, we directly evaluated mucosal CD8+ T cell-mediated immunity using ex vivo cytotoxic T lymphocyte (CTL) assay and MHC class I tetramer staining method in iliac lymph node (LN) and vaginal tracts of mice immunized mucosally with several prime-boost protocols after genital infection of herpes simplex virus type 1 (HSV-1). Ex vivo CTL activity in iliac LN of infected mice was evaluated at 3-day post-infection without in vitro 5-day stimulation. Iliac LN of mice immunized with recombinant viral vaccine-priming and DNA vaccine-boosting protocol showed more potent CTL activity than those of other groups. Such ex vivo CTL activity was consistent with mucosal gB498-505 (SSIEFARL)-specific CD8+ T cell number of vaginal tract determined by MHC class I (H-2b) tetramer containing immunodominant peptide. Furthermore, the number of mucosal SSIEFARL-specific CD8+ T cells recruited into infected genital tracts appeared to decide the protective outcome against genital infection of virulent HSV-1. These results support that mucosal CD8+ T cells are principal mediators for the protection against genital infection of HSV-1.