Protein S functions as a cofactor with activated protein C in the down-regulation of the blood coagulation cascade. In vitro studies have historically produced conflicting data with regard to the extent of various protein S activity in clotting assays which typically involve adding CaCl(2) to initiate reactions. We report here that protein S reversibly self-associates in the absence of Ca(2+). Sedimentation experiments showed a transition in sedimentation velocity from 7.2 to 4.2 S with a transition midpoint (T(m)) of 0.42 mM Ca(2+) for intact protein S. Studies of thrombin cleaved (Arg(70)) protein S revealed similar results with a transition in sedimentation velocity from 7.9 to 4.4 S with a T(m) of 0.42 mM Ca(2+). This transition is reversible with the addition of 10 mM EDTA. Sedimentation equilibrium data suggest at a minimum, a monomer-dimer-trimer association. Sedimentation velocity experiments were also performed on mixtures of protein S and prothrombin which showed no heterodimer formation in either Ca(2+) or EDTA solutions. These data suggest that previous interpretations of protein S structure and function may have been confounded by the self-associative behavior of protein S in non-Ca(2+) solutions.