HLA-DM catalyzes the release of MHC class II–associated invariant chain–derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell–mediated antigen presentation to antigens internalized by membrane immunoglobulin.