The enzymes that degrade proteins to peptides for presentation on MHC class II molecules are poorly understood. The cysteinal lysosomal proteases, cathepsin L (CL) and cathepsin S (CS), have been shown to process invariant chain, thereby facilitating MHC class II maturation. However, their role in Ag processing is not established. To examine this issue, we generated embryonic fibroblast lines that express CL, CS, or neither. Expression of CL Cl or CS mediates efficient degradation of invariant chain as expected. Ag presentation was evaluated using T cell hybridoma assays as well as mass spectroscopic analysis of peptides eluted from MHC class II molecules. Interestingly, we found that the majority of peptides are presented regardless of CL or CS expression, although these proteases often alter the relative levels of the peptides. However, for a subset of Ags, epitope generation is critically regulated by CL or CS. This result suggests that these cysteinal proteases participate in Ag processing and generate qualitative and quantitative differences in the peptide repertoires displayed by MHC class II molecules.