Cytotoxic T lymphocyte (CTL) responses to vaccinia virus (VV) were studied in human subjects receiving smallpox vaccine by dermal scarification. After in vitro restimulation with VV, peripheral blood mononuclear cells (PBMCs) from three of six vaccinated subjects killed virus-infected target cells. VV-specific, HLA-restricted CTL activity was mediated primarily by CD8+ cells, although low levels of lytic activity by CD4+ cells were observed in some experiments. Two of the three responders had no history of exposure to VV prior to vaccination, whereas all three non-responders were vaccinated against smallpox during childhood. PBMCs from an additional three subjects who had received smallpox vaccine at least 20 years previously were negative for CTL activity. These data suggest that the duration of memory CTL populations against VV in the peripheral blood is limited, and that pre-existing immunity to VV may interfere with boosting of the CTL response upon revaccination.