The endopeptidases (EPs) in wheat endosperm during seed germination and subsequent seedling growth were characterized by gradient-polyacrylamide gel electrophoresis with gelatin copolymerized into the gel. Four cysteine EPs (EP1, EP2, EP3 and EP4) were detected in wheat endosperm during the 7 d growth after seed imbibition. The results also showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0 degrees C. All EPs were completely inhibited by 25 micromol/L E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.