An extracellular protease of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine protease. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. When the enzyme was chemically modified with PMSF, which specifically reacted with serine residue on the enzyme, the activity was eliminated. The endopeptidase activity was inhibited by the modifier which chemically modified carboxyl group of aspartate and glutamate. PLP, which would modify lysine residue, did not affect the endopepetidase activity to a greater extent. This demonstrates that serine and aspartate (or glutamate) residues of enzyme would participate in a important function of the endopeptidase activity.