Although the binding sites of MHC class II molecules can accommodate longer ligands, peptides of 15 to 20 residues are the primary form of processed Ag recovered from class II dimers isolated from living cells. These peptides are derived from intact Ags by proteolysis in endocytic organelles, where binding to class II dimers also occurs. Whether generation of these short peptides typically precedes association with class II molecules, or whether class II molecules initially bind to unfolded proteins or large protein fragments, followed by degradation of the unprotected regions, remains unknown. Here we report the identification of an SDS-stable, long-lived, 120-kDa complex composed of two class II dimers bound to a common large Ag fragment. This complex is produced within the endocytic pathway from newly synthesized MHC class II molecules following exposure of the cells to exogenous hen egg lysozyme. These data suggest that a major pathway of Ag processing involves the initial binding of class II heterodimers to large protein substrates upon exposure of regions with suitable motifs, followed by cleavage and/or trimming of the exposed protein around this bound region. This sequence of events during Ag processing may provide a partial molecular explanation for the immunodominance of certain determinants in protein Ags.