We identified a modified vaccinia virus Ankara (MVA)-peptide recognized by T cells of a vaccinated patient, applying a modified expression-based procedure. CD8(+) T cells reactive with MVA were expanded from peripheral blood of a patient after MVA-vaccination. The restricting HLA-elements and the antigen were determined applying stably HLA-class I-transfected K562 cells that were either infected wit MVA or transfected with a panel of MVA open reading frames (ORF). The T cells recognized the ORF 28R (K7R) gene product in association with HLA-B*1501 and the peptide-coding region was localized applying truncated in vitro transcribed mRNA. The 9-mer peptide encoded (ORF 28(25-33), SIIDLIDEY) was identified that was also recognized by ex vivo CD8(+) T cells from the patient and further healthy individuals.