Protein antigens require limited proteolytic processing to generate peptides for binding to class II MHC molecules, but the proteases and processing sites involved are largely unknown. Here we analyze the effect of eliminating the three major asparagine endopeptidase (AEP)-processing sites in the microbial antigen tetanus toxin C fragment. The mutant antigen is highly resistant to proteolysis by AEP and crude lysosomal extracts and is dramatically impaired in its ability to be processed and presented to T cells. Remarkably, processing at a single asparagine residue (1219) is obligatory for optimal presentation of many T cell epitopes in this antigen. These studies demonstrate that cleavage at a single processing site can be crucial for effective antigen presentation.