Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (<5% of total protein S) binds with high affinity to phospholipid bilayers (K(d) < 1 nM). The remaining protein S (>95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (<5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.