Evidence is presented that suggests a role for transmembrane domain interactions in the assembly of class II major histocompatibility complex (MHC) molecules. Mutations in the transmembrane domains of the class II MHC α or β chains resulted in proteins that did not generate complexes recognized by conformation-dependent antibodies and that were largely retained in the endoplasmic reticulum. Insertion of the α and β transmembrane domains into other proteins allowed the chimeric proteins to assemble, suggesting a direct interaction of the α and β transmembrane domains. The interactions were mediated by a structural motif involving several glycine residues on the same face of a putative α helix.