Efficient loading of major histocompatibility complex class II molecules with peptides requires the invariant chain (Ii) and the class II-like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii-/-M-/-). Antigen presenting cells (APCs) from Ii-/-M-/- mice, as compared with APCs from Ii-/- mice, exhibit a significant reduction in their ability to present self-peptides to a panel of class II I-Ab-restricted T cells. As a consequence of this defect in the loading of self peptides, CD4(+) thymocyte development is profoundly impaired in Ii-/-M-/- mice, resulting in a peripheral CD4(+) T cell population with low levels of T cell receptor expression. These findings are consistent with the idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.