Two serine proteases from the viscera of deep-sea fish, black oreo dory (Allocyttus niger),were purified by hydrophobic, affinity, and cation exchange chromatography. They were designated as chymotrypsins on the basis of substrate specificity and susceptibility to inhibitors. The pH optima of chymotrypsin I and II were 8.6 and 10, respectively. Chymotrypsin II retained a remarkable 80% activity at pH 12.5. Thermal stability of both enzymes was enhanced in the presence of calcium ions. Both chymotrypsins were inhibited by high concentrations of substrate Suc-AAPF-NA.