Intracellular localization and routing of MHC class II molecules and the associated invariant chain (Ii) were studied using rat-2 cells and HeLa cells stably transfected with the murine class II genes A alpha kA beta k (Ak) alone or supertransfected with human Ii or a truncated delta 20Ii lacking the endosomal sorting signal within Ii. We show that Ii and delta 20Ii are able to bind the class II molecules and replace the highly homologous murine Ii in class II sorting. Expression of transfected Ii or delta 20Ii also result in more efficient transport of class II molecules from the ER. There are, however, cell type specific differences in the intracellular routing of the class II molecules. The endosomal sorting signal within Ii is required to target class II molecules to endosomes in HeLa cells, whereas class II molecules alone enter endosomes in rat-2 cells. Class II molecules cannot direct the associated delta 20Ii to endosomes, suggesting that they follow a direct route to the cell surface and the majority of class II may enter endosomes by internalization. This is also supported by the long lived presence of class II molecules in endosomes after arresting protein transport with cycloheximide or brefeldin A. Class II molecules expressed alone or together with Ii are located in the whole endosomal pathway in rat-2 cells. However, full length Ii is required to target the class II molecules to vesicles that are more chloroquine sensitive than vesicles containing class II molecules alone. Our results thus indicate that Ii is responsible for endosomal sorting of class II molecules in HeLa cells, whereas in rat-2 cells the presence of Ii leads to an altered endosomal distribution.