Abstract Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated anti-tyrosinase T-cell populations as well as ex vivo-CD8 + lymphocytes from HCMV-seropositive donors. APC electroporated with clonal mRNA were efficient inducers of spot formation by antigen-experienced CD8 + T cells. They were equivalent to peptide-loaded targets. mRNA electroporation did not induce non-specific spot formation. While the use of autologous mRNA-electroporated DC can uncover the complete individual T-cell response towards an antigen, mRNA-electroporated K562 cells stably transfected with single HLA class I alleles help to detect CD8 + T-cell responses restricted by single HLA class I molecules.