AbstractPeptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory moleculeH2‐M, which helps to exchange the invariant chain‐derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2‐O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2‐M and selectively expressed in B cells. Using stable H2‐O ribozyme‐antisense transfectants, H2‐O overexpressing murine B cell lines, and H2‐O‐transgenic mice, we investigated the effects of H2‐O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levelsof H2‐O in the antigen‐presenting B cells. Thus, increased H2‐O expression downmodulated, whereas reduced H2‐O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2‐O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2‐O levels. Theintracellular location of H2‐O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2‐O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2‐O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.