Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen-presenting cells must be broken down into antigenic peptides. Second, class II molecules are synthesized with their peptide-binding site blocked by invariant chain (Ii), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified mice deficient in single lysosomal proteases has allowed us to determine their role in these processes. Cathepsins (Cat) B and D, previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immune responses and for T-cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co-exist in the same antigen-presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications.