HLA class II antigens are heterodimeric cell surface glycoproteins that interact with antigenic peptides to form complexes recognizable by CD4-positive T cells. During their biosynthesis, class II antigens are retained in a post-Golgi compartment in association with the invariant chain, which dissociates before class II cell surface expression. To address whether the invariant chain mediates this post-Golgi retention, its transport and assembly were examined in cells that do not express HLA class II antigens. Pulse-chase analysis and endoglycosidase digestions showed that very little invariant chain proceeded as far as the trans-Golgi in class II-negative cell lines. Immunofluorescence studies suggested that in these cells the invariant chain is sequestered in the RER. Gel filtration and cross-linking data showed that RER-localized invariant chain is present as trimers or aggregated trimers. Multimerization is mediated by lumenal interactions; a proteolytic fragment of the invariant chain corresponding to the lumenal domain remained trimeric as determined by cross-linking analysis. Similar transport and structural characteristics were observed for a pool of excess invariant chain in class II-positive cells, suggesting that an excess of invariant chain in the ER may be important for class II antigen function. These results have important implications for the transport of cellular proteins in general and for the role of the invariant chain in class II antigen biosynthesis.