A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 • C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn 3 , Leu 6 , His 10-Leu 11 , Ala 14 , Glu 21 , after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P 1 position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a K m values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool.