In this report we describe a novel chymotrypsin-like serine protease produced by Streptomyces griseus. The enzyme has been tentatively named S. griseus protease C (SGPC). The gene encoding the enzyme (sprC) was identified and isolated on the basis of its homology to the previously characterized S. griseus protease B (SGPB). The sprC gene encodes a 457-amino acid prepro-mature protein of which only the 255 carboxyl-terminal amino acids are present in the mature enzyme. Mature SGPC contains two distinct domains connected by a 19-amino acid linker region rich in threonines and prolines. While the amino-terminal domain is homologous to S. griseus proteases A, B, and E and the alpha-lytic protease of Lysobacter enzymogenes, the carboxyl-terminal domain is not homologous with any known protease. However, the carboxyl-terminal domain shares extensive homology with chitin-binding domains of Bacillus circulans chitinases A1 and D, suggesting that the enzyme is specialized for the degradation of chitin-linked proteins. Recombinant expression and preliminary characterization of the catalytic properties of the enzyme are also reported. The primary specificity of SGPC is similar to that of SGPB; both enzymes preferentially cleave peptide bonds following large hydrophobic side chains.