Current ideas about DM actions have been strongly influenced by studies of mutant strains expressing the H-2 b haplotype. To evaluate DM contributions to class II activities in BALB/c mice, we generated a novel mutation at the DMa locus via embryonic stem cell technology. Unlike long-lived A b/class II-associated invariant chain-derived peptide (CLIP) complexes, mature A d and E d molecules are loosely occupied by class II-associated invariant chain-derived peptide and are SDS unstable. BALB/c DM mutants weakly express BP107 conformational epitopes and toxic shock syndrome toxin-1 superantigen-binding capabilities, consistent with partial occupancy by wild-type ligands. Near normal numbers of mature CD4+ T cells fail to undergo superantigen-mediated negative selection, as judged by TCR Vβ usage. Ag presentation assays reveal consistent differences for A d-and E d-restricted T cells. Indeed, the mutation leads to decreased peptide capture by A d molecules, and in striking contrast causes enhanced peptide loading by E d molecules. Thus, DM requirements differ for class II structural variants coexpressed under physiological conditions in the intact animal.