Protein S is a natural anticoagulant present in the plasma that serves as a cofactor for activated protein C. Patients deficient in protein S are subject to recurrent venous thrombotic disease. Protein S deficiency differs from other plasma protein deficiencies in that deficient patients often have normal or only mildly reduced levels of protein S in their plasma as detected by conventional immunologic methods but have markedly reduced functional protein S levels. This apparent discrepancy is due to the presence of two forms of protein S in plasma. The protein S is present free and in a complex with C4b-binding protein. The free form is functionally active, whereas the bound form is not. Examination by crossed immunoelectrophoresis of 31 functionally protein S-deficient individuals from seven families reveals that 29 of the 31 have all or most of their protein S complexed to C4b-binding protein with little or no free protein and have correspondingly low levels of protein S functional activity (type I deficiency). Two related protein S-deficient individuals show a different type of distribution with little or no protein S, either bound or free (type II deficiency). The detection and classification of protein S-deficient individuals requires the application of both a functional assay and an assessment of protein S distribution between bound and free forms.