Invariant (Ii) chain and DM functions are required at distinct stages during class II maturation to promote occupancy by diverse peptide ligands. The class II molecules expressed by mutant mouse strains lacking Ii chain or DM activities display discrete structural and functional abnormalities. The present report describes the cellular and biochemical characteristics of Ii-DM- doubly deficient mice. As for Ii chain mutants, their mature Aalphab Abetab dimers similarly exhibit reduced mobilities in SDS-PAGE, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. Additionally, the present experiments demonstrate that the production of floppy Aalphab Abetab dimers is TAP independent. In comparison with Ii chain mutants, Ii-DM- doubly deficient cell populations exhibit increased peptide binding activities and consistently greater presentation abilities in T cell stimulation assays. These functional differences appear to reflect higher class II surface expression associated with their increased representation of B lymphocytes. We also observe defective B cell maturation in mice lacking Ii chain or DM expression, and interestingly, B cell development appears more severely compromised in Ii-DM- double mutants. These mutant mice lacking both Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation under normal physiological conditions in the intact animal.