Dendritic cells (DC) initiatiate immunity and maintain tolerance. They internalize exogenous antigen and convert it into immunogenic peptides by lysosomal proteolytic degradation, ultimately followed by presentation to CD4 T cells. Monocyte-derived DC (MO-DC) generated in vitro with GM-CSF and IL-4 serve as prototype DC to analyse the cellular biology and biochemistry of DC. However, different types of primary DC, whose functional role in vivo and relationship to MO-DC generated in vitro is unclear, reside in human tissue as well as peripheral blood. The composition of lysosomal proteases in these primary human DC1 and DC2-cells and the way they handle a clinically relevant antigen are unknown, and there is no comparison of the lysosomal processing of antigen by these primary DC to that in primary human B cells or MO-DC generated ex vivo. We have isolated human peripheral blood (PB) DC1 and DC2 cells as well as primary B lymphocytes by magnetic separation and isolated lysosomal compartments from these cells, as well as from MO-DC. Expression and activity of endocytic proteases were assessed by western blot and active site-restricted affinity labelling using a synthetic probe that selectively binds to the active centre of cysteine proteases and allows a simultaneous semiquantitaive assessment and identification of multiple active protease species. In this analysis, PB-DC1 and DC2-cells lacked significant active Cathepsins (Cat) S, L and B as well as asparagine-specific endoprotease AEP, the major enzymes involved in antigen processing in the MHC II-compartment. Surprisingly, lysosomal extracts from PB-DC1 were by far more effective than MO-DC in processing the muliple sclerosis-associated autoantigen myelin basic protein (MBP) in vitro. When analyzed on a molecular scale using mass spectrometry, MBP processing was dominated by CatS, CatD and AEP in MO-DC, as expected, similar to B-lymphoblastoid cells (BLC). PB-DC, however, did not generate proteolytic processing intermediates indicative of CatS or AEP activity but showed the same pattern as primary B-lymphocyte-derived lysosomes, i.e. processing was performed by two cleavage sites that can be reproduced by purified CatG in vitro, suggesting a CatG like dominant lysosomal protease. While active CatG was present in primary human B cells, PB-DC1 cells lacked CatG protein by western blot, suggesting the presence of an as yet unknown dominant endoprotease with CatG-like activity in PB-DC1. By cleaving MBP after pos F90 and F114, this protease directly eliminates the integrity of the major immunodominant MBP epitope MBP85-99. This might lead to poor presentation of this epitope to regulatory T cells resulting in inefficient silencing of MBP-autoreactive T cells during the development of autoimmunity. Our results emphasize the need to apply state-of-the-art biochemical tools to primary human types of APC for the understanding of antigen processing and the rational design of tolerogenic or immunotherapy approaches towards human malignant and autoimmune disorders.