This study measured the ability of a standard smallpox vaccine, given by scarification (by bifurcated needle), to induce primary human vaccinia virus-specific cytotoxic and interferon (IFN)-γ-producing T lymphocyte responses. Because protection against smallpox may be mediated in part by T cell memory responses induced by vaccination, an analysis of the induction of primary human cytotoxic T lymphocytes (CTL) and IFN-γ-producing T cell responses was performed. Although smallpox is no longer an epidemic threat under natural conditions, vaccination is still recommended for persons working with vaccinia viruses in the laboratory and for those who may be at risk from the potential use of smallpox virus as a bioterrorism agent. The results demonstrate that smallpox vaccine given by bifurcated needle induces strong vaccinia virus-specific CD8+ CTL and IFN-γ-producing T cell responses and provide baseline information useful for planning the immunologic assessment of future smallpox vaccines.