Peripheral T lymphocytes can be classified into two groups: naive and memory T cells. The focus of this study was to examine the duration of T-cell memory in humans. Vaccinia virus replicates in the cytoplasm of infected cells and is not thought to persist or become latent after the acute phase of infection. We identified long-lived vaccinia virus-specific memory cytotoxic T cells in adults who had been immunized against smallpox as children. Initially, we detected vaccinia virus-specific T cells in peripheral blood mononuclear cells while screening for human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV-1-seropositive subjects. These individuals had not had contact with vaccinia virus since their primary immunization in early childhood. Several vaccinia virus-specific CD4+ T-cell clones were derived from these donors and characterized. Healthy, HIV-1-seronegative donors who had been immunized against smallpox many (35 to 50) years earlier were also screened for vaccinia virus-specific T-cell immunity. We found significant CD8+ and CD4+ cytotoxic T-cell responses to vaccinia virus after in vitro stimulation, indicating that these memory cells are maintained in vivo for many years. The peripheral blood mononuclear cells of young adults with no history of immunization against smallpox did not develop vaccinia virus-specific T-cell responses after in vitro stimulation. Precursor frequency analysis of the vaccinia virus-specific memory CD4+ T cells from a donor immunized with vaccinia virus 35 years earlier revealed a frequency of 1 in 65,920 CD4+ T cells. We concluded that specific vaccinia virus T-cell immunity can persist for up to 50 years after immunization against smallpox in childhood in the presumed absence of exposure to vaccinia virus.