A pronounced Ca2+-dependent protease in lysed platelets was detected by the hydrolysis of an added substrate, azocasein. This protease also catalyzed the cleavage of endogenous platelet polypeptides which was demonstrated by the disappearance of four high molecular weight polypeptides (greater than 200,000) either by adding Ca2+ to lysed platelets or by increasing the cytoplasmic Ca2+ within intact platelets with the ionophore A 23187. In both cases, lower molecular weight cleavage products were clearly identified. The caseinolytic activity as well as the cleavage of the high molecular weight polypeptides was inhibited by sulfhydryl reagents, but not by reagents reacting with trypsin-like proteases. Because of the similarity between this Ca2+-dependent protease and myofibrillar Ca2+-dependent protease (CAF), we have termed the former "platelet CAF".