Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.