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Presentation of material derived from pathogenic organisms to the immune system requires uptake of antigens into antigen presenting cells, processing into peptide fragments and loading of the resulting fragments onto major histocompatibility complex (MHC) class II molecules. MHC class II-restricted antigen presentation involves both the biosynthetic as well as the endocytic pathway of antigen-presenting cells. In recent years, the general mechanisms that govern these processes have been delineated, and specialized organelles have been characterized in which processing and loading of antigens takes place. Here, we review the work that has led to the characterization of these MHC class II compartments, and describe the use of organelle electrophoresis and two-dimensional gel electrophoresis to analyze the molecular composition of the different subcellular organelles involved in MHC class II-restricted antigen presentation as well as in antigen uptake.