Directed dissemination of Ag-specific CD8(+) T cells to infected organs or cancerous tissues is a prerequisite for optimal immunotherapy. Ag-specific CD8(+) T cells were quantitated in systemic and mucosal tissues after nasal, rectal, or cutaneous immunization with CTL epitope peptide and the adjuvant cholera toxin (CT). Mucosal and cutaneous immunization induced Ag-specific CD8(+) lymphocytes that were detectable in both mucosal and systemic compartments, suggesting a less strict distribution pattern than that known for B cells. However, optimal localization, activation and phenotype of these cells correlated with the route of immunization. In accordance with this observation, protection against a mucosal challenge with a virus expressing the CTL epitope was superior in mucosally-immunized animals.