V8 protease, a member of the glutamyl endopeptidase I family, ofStaphy-lococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coliexpression system for GluV8, as well as its homologue fromStaphylococcus epidermidis(GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated.