Digestive proteases from the digestive tract of sea cucumber (Stichopus japonicus) have been characterized. There exist acidic pro-teases with optimum activities at pH 2.0 and 5.0, and alkaline proteases with optimal activities at pH 8.0 and 13.5, in the digestive tract of sea cucumber. The result of SDS-PAGE with or without protease inhibitors at a series of pH ranges confirmed the existence of at least three proteases with molecular weights of 20.6, 39.1 and 114.1 kDa. The high alkaline protease (HAP), with an optimum pH of 13.5, was purified using a combination of ion exchange and gel filtration chromatography. The purified HAP was then char-acterized in detail. The results showed that the molecular weight of the HAP is 20.6 kDa, and that it has relatively good thermal and pH stability with an optimum pH of 13.5 and optimum temperature of 37 C. Therefore, it could be well applied in many fields to meet special needs, especially under extremely alkaline conditions. According to the inhibition profiles obtained with numerous spe-cific protease inhibitors and the renaturing effect of Cu2+ on the EDTA-denatured HAP, the purified HAP was defined to be a metallo-protease containing Cu2+ with serine protease-like activity.