Human leukocyte antigen DM (HLA-DM) molecules are structurally related to classical MHC class II molecules and reside in the lysosome-like compartment where class II-restricted antigen processing is thought to occur. Mutant cell lines lacking HLA-DM are defective in antigen processing and accumulate class II molecules associated with a nested set of invariant chain-derived peptides (class II-associated invariant chain peptides, CLIP). Here we show that HLA-DM catalyzes the dissociation of CLIP from MHC class II-CLIP complexes in vitro and facilitates the binding of antigenic peptides. The reaction has an acidic pH optimum, consistent with its occurrence in a lysosome-like compartment in vivo. Antibody blocking experiments suggest that a transient interaction between HLA-DM and the MHC class II-CLIP complex is required.