The molecular characterization of unknown naturally presented major histocompatibility complex (MHC) class II glycopeptides carrying complex glycans has so far not been achieved, reflecting the different fragmentation characteristics of sugars and peptides in mass spectrometric analysis. Human leukocyte antigen (HLA)‐DR‐bound peptides were isolated by affinity purification, separated via high performance liquid chromatography and analyzed by matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry. We were able to identify two naturally processed MHC class II ligands, CD53_122–136 and CD53_121–136, carrying complex N‐linked glycan side chains by a combination of in‐source and collision‐induced fragmentation on a quadrupole time‐of‐flight tandem mass spectrometer. Copyright © 2005 John Wiley & Sons, Ltd.