Calphobindin-II (CPB-II, annexin VI), is a calcium dependent phospholipid binding protein that can be classified as a member of the annexin family. The phospholipid-binding properties of CPB-II were investigated by measuring the binding constants of [125I]-CPB-II using phospholipid vesicles consisting of 80% phosphatidylcholine and 20% phosphatidylserine. A dissociation constant (Kd) of CPB-II with the phospholipid vesicles was determined to be 0.2 to 0.3 nM in the presence of Ca2+ ranging from 0.3 to 30 mM. The number of CPB-II capable of binding to the phospholipid vesicles at 0.3 mM Ca2+ decreased to about 1/2 in the presence of Ca2+ of more than 1 mM. Prothrombin and factor X were effective in competing with the binding of CPB-II to the phospholipid vesicles, although their affinities were lower by two or three orders of magnitude than that of unlabeled CPB-II at 30 nM Ca2+. Competitive effects of CPB-II, calphobindin-I (CPB-I, annexin V) and calphobindin-III (CPB-III, annexin III) on binding of [125I]-CPB-II to phospholipid vesicles, were similarly observed.