Abstract Although the number of defined T cell epitopes of clinically relevant antigens is constantly increasing, there is still an enormous need to identify further peptides, processed from new antigens or presented by rare HLA molecules, respectively. Here we introduce a novel two-step approach for the rapid identification of T cell epitopes. It was established in the CMV infection model. From the peripheral blood of healthy donors sharing HLA-A1 according to HLA serotyping we isolated CD8 + T lymphocytes and generated dendritic cells (DCs). DCs were electroporated with CMV pp65 mRNA and tested for recognition by autologous CD8 + T lymphocytes in IFN-γ ELISPOT assays. In all 10 CMV-seropositive donors, CMV pp65-specific CD8 + T cells were readily detectable ex vivo. In 7 of them the response was at least in part restricted by HLA-A1.1 as verified in IFN-γ ELISPOT assays with pp65 mRNA-electroporated K562 cells stably transfected with HLA-A*0101 (K562/A*0101). In a subsequent step various 3′-deleted pp65 RNA fragments were rapidly generated by in vitro transcription of plasmid DNA-templates linearized with restriction enzymes at different sites within the pp65-coding sequence. Polyadenylated mRNA fragments were then electroporated into K562/A*0101 cells and tested for recognition by ex vivo CD8 + T cells in IFN-γ ELISPOT assays. We thereby identified a 76 bp-long sequence as target of the HLA-A*0101-associated pp65-specific T cell response. From this region, 10 peptides predicted by current algorithms were synthesized and tested for recognition. Peptide pp65 364–373 (previously identified by a reverse immunology approach by [Hebart, H., Daginik, S., Stevanovic, S., Grigoleit, U., Dobler, A., Baur, M., Rauser, G., Sinzger, C., Jahn, G., Loeffler, J., Kanz, L., Rammensee, H. G., Einsele, H., 2002. Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses by interferon-gamma-enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation, Blood 99, 3830.]) was positively tested and found to be the dominant target epitope of the HLA-A1-restricted anti pp65 T cell response in all donors. We conclude that (i) the use of HLA-transfected K562 cells allows to dissect antigen-specific T cell responses to partial responses associated with defined HLA class I alleles and (ii) transfection of in vitro transcribed RNA fragments allows to identify immunogenic regions of a given antigen. The latter technique bypasses the need of prior cloning and sequencing of cDNA fragments, reduces the number of synthetic peptides to be tested and thus saves both costs and time.