The Ca(2+)-phospholipid binding proteins in human placental tissue were investigated with the binding of a placental EDTA extract to liposomes composed of placental phospholipids. A new Ca(2+)-dependent phospholipid-binding protein different from calphobindin I (CPB I) and calphobindin II (CPB II) was isolated from the EDTA extract, and the purification procedure of this protein was established. The yield of the purified protein was about 1.2 mg from one placenta. The protein prolonged the clotting time of normal plasma when coagulation was induced by tissue factor and ellagic acid. This protein had an apparent molecular weight of 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and its isoelectric point was 5.8. Because of its ability to bind phospholipids in the presence of Ca2+, this protein was designated as calphobindin III (CPB III).