Adoptive T‐cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen‐specific T cells. However, the in vitro generation of adequate numbers of functional antigen‐specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen‐specific T cells for adoptive T‐cell transfer. We have developed a new artificial antigen‐presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide‐β2‐microglobulin–MHC fusion construct (single‐chain aAPC) ensuring presentation of the peptide–MHC complex of interest. Using this artificial antigen‐presenting cell, we could generate up to 9·2 × 10⁸ antigen‐specific cytotoxic CD8⁺ T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single‐chain aAPC with autologous monocyte‐derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen‐specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single‐chain aAPC represent a promising option for the generation of antigen‐specific CD8⁺ T cells, which could be used for adoptive T‐cell therapy.