HLA-DR antigens were isolated from B lymphoblastoid cells labeled with either [3H]- or [14C]lysine. Double-label comparisons of the tryptic peptides by microbore ion-exchange chromatography led to the following conclusions: The heavy and light chains share few peptides, one of which is free lysine that is liberated from the polypeptide chains by trypsin. The heavy chains of different HLA-DR allospecificities are virtually identical in all cell lines examined. In contrast, the light chains of different HLA-DR allospecificities are nonidentical and, thus, must bear the alloantigenic determinant.