We previously demonstrated that vaccinia virus (VV)-specific CD4+ cytolytic T cells can persist for> 50 years after immunization against smallpox in the absence of re-exposure to VV. Nevertheless, there have been few studies focusing on CD4+ T cell responses to smallpox vaccination. To ensure successful vaccination, a candidate vaccine should contain immunodominant CD4+ T cell epitopes as well as CD8+ T and B cell epitopes. In the present study, we established cytotoxic CD4+ T cell lines from VV-immune donors, which recognize epitopes in VV proteins D1R and A24R in association with HLA-DR1 Ags. Comparisons of sequences between different members of the poxvirus family show that both epitopes are completely conserved among VV, variola viruses, and most mammalian poxviruses, including monkeypox, cowpox, and ectromelia. The CD4+ T cell lines lysed VV-infected, Ag-and peptide-pulsed targets, and the lysis was inhibited by concanamycin A. We also detected these peptide-specific cytolytic and IFN-γ-producing CD4+ T cells in short-term bulk cultures of PBMC from each of the three VV-immune donors tested. These are the first VV-specific CD4+ T cell epitopes identified in humans restricted by one of the most common MHC class II molecules, HLA-DR1, and this information may be useful in analyzing CD4+ T cell responses to pre-existing or new generation VV vaccines against smallpox.