The calcium-binding properties of the coagulation cofactor, Factor V, have been investigated using atomic absorption spectrophotometry, equilibrium dialysis, and chemical exchange experiments. Two classes of binding sites have been observed: one site containing a single Ca2+ ion bound with an unusually high affinity (Kd less than 10(-8) M), and a second site in which 2 mol of calcium are bound/mol of Factor V with an association constant Ka = 1.7 +/- 0.52 x 10(4) M-1 (Kd = 5.9 +/- 1.9 x 10(-5) M). The binding of the dissociable Ca2+ ions is apparently noncooperative. The single high affinity Ca2+ ion may be removed readily by EDTA under native conditions with an immediate loss of Factor V activity, and the activity of the Factor V can be restored by the addition of a molar excess of calcium. The high affinity Ca2+ ion will exchange with radiolabeled calcium in solution, and Factor V labeled in this way was used to provide an independent measure of the stoichiometry of this high affinity binding site, and to observe directly the interaction of calcium in this site with EDTA. In addition, elemental analyses of solid lyophilized samples of Factor V revealed that no transition metals are present and that no phosphorus (or phospholipid) is retained by the Factor V after purification.