The immunogenicity of recombinant modified vaccinia Ankara, a highly attenuated vaccinia virus, expressing influenza nucleoprotein (MVA-NP) and HIV-1 gag (MVA-gag) was investigated. Restimulation of peripheral blood lymphocytes of healthy subjects with MVA-NP led to expansion of CTL with specificity for known NP epitopes. These CTL efficiently lysed NP peptide-pulsed targets and released interferon-gamma (IFN-gamma) on contact with epitope peptide. MVA-NP-stimulated CTL specific for the HLA-B8 epitope, NP380-88, stained with a tetrameric complex of HLA-B8 refolded with the NP380-88 peptide and anti-CD8 antibody on flow cytometry. CTL were also elicited from two HIV-1 seropositive donors by restimulation with MVA-HIV-1 gag and showed specificity for immunodominant gag epitopes. These data indicate that restimulation of human CTL with recombinant MVA is effective and suggest that MVA will elicit CTL responses in humans in vivo.