Shedaoenase, a serine protease, was isolated from the venom of Agkistrodon shedaoenthesis Zhao with an apparent molecular mass of 36 kDa. It was purified by affinity chromatography on arginine Sepharose 4B column and anion exchange on Mono Q fast protein liquid chromatography. Shedaoenase preferentially cleaved the Aalpha-chain of human fibrinogen and slowly digested the Bbeta-chain. It also showed arginyl esterase activity using Nalpha-benzoyl-L-arginine ethyl ester as a substrate, and some synthetic chromogentic substrates, such as Chromozym PL, S-2266, and S-2160, could also be hydrolyzed. The enzyme activity of shedaoenase could be completely inhibited by phenylmethylsulphonylfluoride and could be little inhibited by the chelating reagent EDTA. The N-terminal sequence of shedaoenase was determined, and its full-length cDNA encoding a protein of 238 amino acid residues was cloned by reverse transcription-polymerase chain reaction from the total mRNA extracted from the snake venom gland. The deduced primary sequence of shedaoenase shares significant homology with other snake venom serine proteases.