A 36-kDa protein, which is a component of the membrane skeleton, has been shown to co-localize with spectrin in addition to serving as a major substrate for tyrosine-protein kinases. This protein, which will be referred to as calpactin (for calcium-dependent phospholipid and actin binding protein), was isolated from bovine intestine as the complex with a 10-kilodalton light chain and the Ca2+ binding was analyzed by equilibrium dialysis with 45Ca2+ in the presence or absence of phospholipid. Although Ca2+ binding by calpactin alone was negligible at micromolar free Ca2+, it was greatly enhanced by liposomes containing phosphatidylserine or phosphatidylinositol. A proteolytic derivative of calpactin, termed the "core," which has lost the site of association with the light chain in addition to the site of tyrosine phosphorylation by pp60src, was also found to contain this high affinity phospholipid enhanced Ca2+-binding activity. Scatchard plots reveal that each calpactin monomer or core polypeptide bound 2 Ca2+ ions with a Kd of 4.5 X 10(-6) M at 200 micrograms of phosphatidylserine/ml. Liposome binding experiments confirmed that calpactin as a complex with light chain as well as calpactin monomer or the 33-kDa core interact with phosphatidylserine liposomes in a Ca2+-dependent manner.