HLA-DR molecules associated with class II—associated invariant chain peptides (CLIP) are generated in vivo as an intermediate in class II maturation. Such complexes can be produced in vitro by proteolytic digestion of DR αβ1 complexes, suggesting that CLIP is a residual fragment that remains associated with class II molecules following I chain degradation. In vitro, CLIP dissociation from DR αβ dimers occurs at different rates depending on the allele, and is facilitated by low pH and by detergents containing 8–10 carbon unbranched hydrocarbons, or by primary allphatic amines or carboxylic acids. The accumulation of DR αβCLIP complexes in HLA-DM-negative antigen-processing mutant cells argues that a functionally similar mechanism, dependent on HLA-DM expression, catalyzes in vivo CLIP dissociation and generation of normal class II—peptide complexes.