Pepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trionyx sinensis) by a series of chromatographies on DEAE-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl-L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymosin.