The crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor which strongly inhibits bacterial alkaline proteinases, was determined at 2.3 Å resolution. The subunit (molecular weight, 11, 485) of this dimeric molecule has a unique fold. of polypeptide chain with a five-fold anti-parallel β-sheet structure (about 21% of the 113 amino acid residues) and two small segments of α-helices (about 16%). The region around the apparent reactive site, Met(73)-Val(74), is held tight by a combination of various structural features. The conformation of this region seems to have close similarity to that found in substrate analogues of low molecular weight bound to subtilisin BPN'.