Protein S, a vitamin K-dependent cofactor for activated protein C, exists in normal adult plasma in a free anticoagulantly active form and in an inactive form complexed to C4b-binding protein. Immunologic and functional levels of protein S and C4b-binding protein in plasma were determined for 20 newborn infants and compared with adult normal pooled plasma. Total protein S antigen levels averaged 23%, similar to other vitamin K-dependent plasma proteins. However, the protein S anticoagulant activity was 74% of that of adult normal plasma. This apparent discrepancy of activity to antigen was shown to be due to low or undetectable levels of C4b-binding protein, which results in the presence of most if not all of protein S in its free and active form. The relatively high level of anticoagulantly active protein S in infants may enhance the potential of the protein C pathway, thereby minimizing risks of venous thrombosis in this group.