In this study, we elucidate the FcεRI-mediated Ag uptake and presentation mechanisms of dendritic cells (DC). We found that FcεRI-bound IgE, after polyvalent but not after monovalent ligation, is efficiently internalized into acidic, proteolytic compartments, degraded, and delivered into organelles containing MHC class II, HLA-DM, and lysosomal proteins. To follow the fate of the fragmented ligand, we sought to interfere with invariant chain (Ii) degradation, a process critical for peptide loading of nascent MHC class II molecules. We found DC to express cathepsin (Cat) S, a cysteine protease involved in li processing by B cells. Exposure of DC to a specific, active-site inhibitor of Cat S resulted in the loss of anti-Cat S immunoreactivity, led to the appearance of an N-terminal Ii remnant, and decreased the export of newly synthesized MHC class II to the DC surface. Furthermore, inactivation of Cat S as well as blockade of protein neosynthesis by cycloheximide strongly reduced IgE/FcεRI-mediated Ag presentation by DC. Thus, multimeric ligands of FcεRI, instead of being delivered into a recycling MHC class II pathway, are channeled efficiently into MIIC (MHC class II compartment)-like organelles of DC, in which Cat S-dependent li processing and peptide loading of newly synthesized MHC class II molecules occur. This IgE/FcεRI-dependent signaling pathway in DC may be a particularly effective route for immunization and a promising target for interfering with the early steps of allergen presentation.