A zinc metalloendoprotease from Streptomyces caespitosus (ScNP) is one of the smallest proteases found to date. It consists of a single polypeptide chain of 132 amino acid residues with a molecular weight of about 15 kDa. According to the amino acid sequence, this protease was classified in the Streptomyces small neutral protease family with an aspartate (Asp93) as a metal-binding site. We have determined the cleavage specificities and the optimal conditions for hydrolysis. The amino-terminal amino acid sequences of the hydrolyzed products of an oxidized insulin B-chain showed a high specificity to aromatic residues such as phenylalanine and tyrosine. The enzyme showed maximal activity against azocasein at pH 6.0 and 50 degrees C. ScNP was inhibited by EDTA and 1,10-phenanthroline. Our studies indicated that ScNP is the smallest metalloendoprotease which hydrolyzes with high specificity and this specificity of ScNP was compared with those of ether metalloendoproteases.