Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.