Membrane vesicles from human platelets were prepared by various disruption and isolation techniques reported in the literature to yield fractions of predominantly surface or intracellular membrane origin. ATP + Mg2+-dependent Ca2+ accumulation and the formation of acylphosphate intermediates of the calcium pump(s) were followed in parallel experiments, and the consequences of a limited proteolysis of the membranes examined. In all types of preparations active Ca2+ uptake had both oxalate-sensitive and insensitive fractions and calmodulin had no effect on the rate of Ca2+ uptake. Limited proteolysis by trypsin eliminated oxalate-sensitive Ca2+ uptake while it had no effect on the oxalate-insensitive fraction. The Ca2+-induced EP complex had an apparent molecular mass of 100-110 kDa in all of the preparations, the EP showing a broad or even duplicated line in most autoradiographies. Mild trypsin digestion resulted in the formation of 80-, 55-, and 35-kDa phosphorylated fragments. The 80-kDa fragment corresponded to the limit polypeptide found in the proteolyzed erythrocyte membrane Ca2+ pump, its phosphorylation was stimulated by lanthanum, and it appeared in a different time course than the smaller fragments. The molecular mass and the formation pattern of the latter species corresponded to the tryptic fragments in the sarcoplasmic reticulum Ca2+ pump. Based on these results we suggest that platelet membrane preparations contain two types of Ca2+ pump proteins, one similar to the sarcoplasmic reticulum-type and the other to the erythrocyte-type enzyme.