Transfection of dendritic cells (DCs) with RNA was shown to be effective in the generation of antigen-specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens. To verify this assumption we used DCs, generated from cytomegalovirus (CMV)-negative or -positive donors, that were electroporated with in vitro-transcribed RNA (in vitro transcript, IVT) coding for the CMV pp65 antigen. We found that transfection of DCs with pp65 IVT induces an expansion of polyclonal CD8(+) T lymphocytes that recognize peptide antigens presented on different HLA molecules. These T lymphocytes are able to lyse DCs pulsed with pp65-derived peptides or transfected with the cognate IVT. Furthermore, this approach allowed the identification of immunodominant epitopes presented upon IVT transfection. Interestingly, transfection of DCs with pp65 IVT resulted in the induction of CD4(+)-specific T cells. Cotransfection of DCs with IVTs coding for the CMV antigens pp65 and IE1 elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, cytotoxic T cells could be generated in two of three CMV-negative donors. Finally, functional CMV-specific autologous cytotoxic T lymphocytes were successfully generated from immunosuppressed patients after allogeneic hematopoietic stem cell transplantation.