In B-lymphoblastoid cells, the HLA-DR-associated invariant chain is processed to a form containing O-linked as well as N-linked oligosaccharides. After neuraminidase treatment, the O-linked carbohydrate is susceptible to digestion with an endoglycosidase (endo-beta-N-acetylgalactosaminidase) that cleaves glycans with the structure Gal(beta 1----3)-GalNAc-Ser/Thr, and sialic acid can be added back to this core oligosaccharide by specific sialyltransferases. Treatment of cells with the sodium ionophore monensin markedly affects the post-translational processing of the invariant chain, although that of associated alpha and beta chains is minimally affected. Only a small portion of the N-linked carbohydrate on the invariant chain is processed to an endoglycosidase-H-resistant form. The sialic acid residues normally found on the O-linked glycans are not added, but at least the first residue, GalNAc, is added. In addition to the changes in glycosylation, an intracellular accumulation of HLA-DR antigens also occurs in monensin-treated cells. The accumulation of HLA-DR antigens and the overall slower turnover rates of the alpha, beta, and invariant polypeptides observed after monensin treatment probably reflects the build-up of newly synthesized proteins in Golgi apparatus-derived vacuoles coupled with a decrease in normal degradation in lysosomes.