Measurement of specific cellular immune responses in patients undergoing immunotherapy is difficult. Established approaches, including cytotoxicity (e.g., 51Cr release) and cytokine release assays, require in vitro culturing for several weeks or more of patients' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunological response does not reflect in vivo conditions. To address these disadvantages, we have used an interferon-gamma (IFN-gamma) Elispot assay for detecting peptide-specific CD8(+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of antigen-presenting cells (APCs). Currently available APCs often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I molecules on their surface. We have transfected Drosophila melanogaster S2 cells and the Lepidopteran line Sf9 with the gene encoding human HLA-A2.1. We demonstrate that insect cells expressing a human HLA molecule effectively function as APCs in the IFN-gamma Elispot assay. Initially the feasibility of the assay was assessed using CD8(+) T cells from HLA-A2.1(+) donors with known reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as APCs abrogated background spots, increasing sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptide-specific T cells that could be measured in the Elispot assay without increasing the background. This approach was then used to measure CD8(+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by melanoma cells. Insect cells expressing human HLA molecules provide a standard APC for monitoring CD8(+) T cell responses to tumor and viral peptides during immunotherapy.