We studied the binding of fluorescein-labeled annexin V (placental anticoagulant protein I) to small unilamellar phospholipid vesicles at 0.15 m ionic strength as a function of calcium concentration and membrane phosphatidylserine (PS) content. As the mole percentage of PS in the membrane increased from 10 to 50%, the stoichiometry of binding decreased hyperbolically from 1100 mol phospholipid/mol annexin V to a limiting value of 84 mol/mol for measurements made at 1.2 m m CaCl 2. Over the same range of PS content, K d remained approximately constant at 0.036 ± 0.011 n m. A similar hyperbolic decrease in stoichiometry was observed with vesicles containing 10 or 20% PS when the calcium concentration was increased from 0.4 to 10 m m. Thus, the density of membrane binding sites is strongly dependent on the membrane PS content and calcium concentration. The effect of calcium on annexin V-membrane binding is proposed to be due to the formation of phospholipid-calcium complexes, to which the protein binds, rather than to an allosteric effect of calcium on proteinphospholipid affinity.