ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to strong background spot production by APC-reactive T lymphocytes or have a low antigen presentation capability. Both phenomena can prevent the detection of low frequency T cell responses in PBMC. In search of alternative APC for ELISPOT assays, the human chronic myelogenous leukemia cell line K562 that per se does not express HLA class I and class II molecules on the cell surface was transfected with the HLA-A*0201 gene. Clonal HLA-A*0201-expressing K562 (K562/A*0201) cells were able to process and present endogenously expressed and exogenously loaded melanoma peptide antigens to HLA-A*0201-restricted cytolytic T lymphocyte clones in cytotoxicity and IFN-gamma ELISPOT assays. K562/A*0201 cells were then used as APC in IFN-gamma spot assays to detect ex vivo CD8(+) T lymphocytes responsive to known HLA-A*0201-binding peptide epitopes derived from cytomegalovirus, Epstein-Barr virus, influenza virus and melanoma in PBMC from HLA-A*0201-positive donors. In the majority of cases, peptide-pulsed K562/A*0201 cells were similarly efficient in the ability to visualize single antigen-specific CD8(+) T lymphocytes when compared to T2 cells. However, in contrast to T2, background reactivity of CD8(+) T cells responsive to unpulsed K562/A*0201 was regularly found to be negligible, thereby enhancing the sensitivity of the ELISPOT assay, particularly in donors with strong anti-T2 reactivity. K562 cells transfected with HLA-A*0201 or other HLA genes can serve as standard APC for monitoring T lymphocyte responses against tumor and viral peptide antigens.