In Saccharomyces cerevisiae, the Kex2 endoprotease (Kex2p) is required for the proteolytic maturation of alpha-pheromone and also for the removal of its own pro-region. Kex2p is specific for pairs of basic amino acid residues. Two putative processing sites are present in the pro-region of Kex2p. We have expressed processing site mutants of Kex2p and assayed the production of active Kex2p. Mutations affecting either putative cleavage site do not alter the activity. However, mutations affecting both sites led to a reduction in both Kex2 activity and the amount of protein. These results suggest that removal of Kex2p pro-peptide is required for the production of a stable enzyme and can occur at either processing site.