Invariant chain (Ii) is a nonpolymorphic, non-major histocompatibility complex (MHC)-encoded glycoprotein that rapidly associates with newly synthesized class II MHC alpha and beta chains in the rough endoplasmic reticulum. This oligomerization of Ii, alpha, and beta and their cotransport within the cell led to speculation that Ii was an essential alpha beta transport protein. However, direct tests failed to show an absolute requirement for Ii in class II MHC molecule transport. More recently, it has become clear that different class II alpha beta chain combinations vary greatly in their efficiency of cell-surface expression, based largely on the allelic origin of the alpha and beta amino-terminal regions. Because the previous tests of Ii for a role in class II molecule expression utilized efficiently expressed alpha beta combinations, we have reexamined this question with several haplotype-mismatched murine A alpha and A beta chain combinations of various potentials for cell-surface expression. Using a transient expression assay in Ii-negative COS cells, we find that many inefficiently expressed alpha beta combinations show marked augmentation of surface expression upon cosynthesis of Ii. This effect is absent or minimal with evolutionarily coselected, haplotype-matched chains that give efficient expression alone. Biochemical studies show that at least one component of the Ii effect is an increased egress of already formed alpha beta dimers from the rough endoplasmic reticulum/cis-Golgi. We suggest that these results reflect the interaction of Ii with the peptide-binding domain of the poorly expressed class II molecules, either aiding in maintenance of a transportable conformation or competing with endoplasmic reticulum retention proteins, and thus enhancing movement to the cell surface. These results suggest a complex and variable role for trans-associated alpha and beta chains in the immune responses of MHC heterozygotes and provide a method for examining Ii interaction with class II MHC molecules independent of measurement of peptide presentation to T cells.