CD8⁺ T lymphocytes recognize antigens as short peptides bound to MHC class I molecules. Available methods cannot determine the number and distribution of these ligands on individual cells or detect antigen-presenting cells in tissues. Here we describe a method for eliciting and identifying monoclonal antibodies specific for a particular peptide–MHC class I combination. One such antibody can identify antigen complexes with a limit of detection approaching that of T cells. We used this antibody to determine the number of peptide–class I complexes generated upon viral infection, to identify antigen-presenting cells in cell mixtures, to determine the site of peptide–MHC class I interaction inside cells, and to visualize cells bearing specific peptide–MHC class I complexes after in vivo infection. Similar antibodies may prove useful for diagnostic or therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.