Identification of pathogen-specific T cells has been greatly facilitated by the advent of synthetic peptide-major histocompatibility complex (MHC) tetramers. In many cases, however, specific epitopes have not been defined, necessitating detection methods that function independently of exact peptide-MHC specificity. Lymphocytes acquire surface proteins from antigen-presenting cells (APCs), and we have exploited this phenomenon to develop the T-cell recognition of APCs by protein transfer (TRAP) assay. This method is based on biotinylation and streptavidin-fluorochrome labeling of APCs, followed by subsequent acquisition of this label by antigen-specific T cells. The TRAP procedure detects MHC class I-restricted T cells regardless of their cytokine profiles or peptide-MHC affinities, and provides a versatile tool for monitoring the phenomenon of APC membrane acquisition by antigen-specific T cells.