In a local immune response, the priming and expansion of the antigen-specific T cell population has been thought to largely take place in the draining lymphoid tissue. This model was primarily based on indirect enumeration of antigen-specific T cells by limiting dilution analyses. Here, tetrameric MHC class I complexes were used to evaluate the contribution of different secondary lymphoid organs in a local immune response by following the CD8+ T cell responses against the immunodominant epitopes of influenza A virus and herpes simplex virus-1. Mice were either intranasally infected with influenza A virus and developed pneumonia or were intradermally injected with herpes simplex virus-1. Remarkably, even though these viruses cause a local infection, the spleen of infected animals contains approximately 50-fold more antigen-specific cytotoxic T cells than the draining lymph nodes. Although antigen-specific T cells in spleen appear not to have experienced any recent encounter with antigen, this population is actively dividing, and over time, the formation of a memory T cell population is observed. These data reveal that there is a remarkably large and distinct population of antigen-specific T cells in spleen in the course of a local antigenic challenge. This T cell compartment may not only form the foundation of a memory T cell pool but could also provide a safeguard against systemic spreading of an infection.