Legume species differ in whether or not the 7S globulins stored in seeds undergo proteolytic processing during seed development, while preserving the bicupin structure and trimeric assembly necessary for accumulation and packing into protein storage vacuoles. Two such cleavage sites have been documented for the vicilins in pea cotyledons: one in the linker region between the two cupin domains, and another in an exposed loop in the C-terminal cupin. In this report, we explain the occurrence of vicilin cleavage in developing pea by showing that the storage vacuoles are already acidified before germination, in contrast to soybean and peanut where acidification occurs only after germination. We also show that the two cleavage reactions are catalyzed by two different proteases. The vicilin cleavage at the linker region was inhibited by AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride), indicative of a serine protease. The cleavage in the C-terminal cupin domain was sensitive to the sulfhydryl-reactive reagents p-chloromercuriphenylsulfonate and iodoacetate, but not to E-64 (N-[N-(L-3-transcarboxyirane-2-carbonyl)-l-leucyl]-agmatine), characteristic of the legumain class of cysteine proteases. During seed development, we found the predominant vicilin cleavage in this pea cultivar (Knight) to be at the site in the second cupin domain; but after germination, both sites were cleaved at about the same rate.