The binding of factor Xa to factor Va in the presence of Ca2+ ions and phospholipid is fundamental for the activation of prothrombin to thrombin. Nevertheless, the biochemistry of the intrinsic association between factors Xa and Va is poorly understood. In the present study we have measured the formation of the protein-protein complex in the absence of phospholipid by using analytical ultracentrifugation. Factor Xa or factor Va were respectively modified with a chromophore-peptidyl-chloromethyl ketone or a thiol-specific chromophore, which permitted selective evaluation of the sedimentation of either component by virtue of its unique absorbance properties. Regardless of which protein was labeled, a factor Xa-Va complex (s20,w = 9.8) was formed. The interaction is specific and reversible. In 2 mM Ca2+ and at 20 degrees C, the dissociation constant for the binding of factor Xa to factor Va is 0.8 microM with a 1:1 stoichiometry. The association has multiphasic Ca2+ dependence. At concentrations of Ca2+ below 1 mM or above 2 mM, a weaker protein-protein equilibrium is maintained.