Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish snakehead (Channa argus) by ammonium sulfate fractionation, anion exchange, and gel filtration. Two-dimensional gel electrophoresis and native-PAGE analysis revealed that their molecular masses were 37, 38, and 36 kDa and their isoelectric points 4.8, 4.4, 4.0, respectively. All of the pepsinogens converted into their active form pepsins under pH 2.0 by one-step pathway or stepwise pathway. The three pepsins showed maximal activity at pH 3.0, 3.5, and 3.0 with optimum temperature at 45, 40, and 40 degrees C, respectively, using hemoglobin as substrate. All of the pepsins were completely inhibited by pepstatin A, a typical aspartic proteinase inhibitor. The N-terminal amino acid sequences of the three pepsinogens were determined to the 34th, 25th, and 28th amino acid residues, respectively. Western blot analysis of the three PGs exhibited different immunological reactions.