Mice intranasally inoculated with influenza A/X-31 are protected against a subsequent intracerebral challenge with the neurovirulent influenza A/WSN and this heterotypic protection is mediated by CD8(+) cytotoxic T lymphocytes. We have studied the kinetics of this secondary immune response and found that despite the elimination of replication-competent virus by day 10, we were able to recover activated influenza-specific cytotoxic T lymphocytes (CTLs) that killed freshly ex vivo from the brains of mice for at least 320 d after the intracerebral inoculation. The activated antiviral CTLs expressed high levels of the early activation marker CD69, suggesting continuing TCR signaling despite a lack of viral protein and major histocompatibility complex staining by immunohistochemistry in the brain parenchyma and barely detectable levels of viral nucleic acid by single and two-step reverse transcription PCR. Local persistence of activated lymphocytes may be important for efficient long-term responses to viruses prone to recrudesce in sites of relative immune privilege.