Protein S deficiency has been demonstrated in 5 members from the same family with a history of severe recurrent venous thrombosis over three generations. The propositus, a 16 year old female, had a first spontaneous thrombotic episode at age 15. Phlebography revealed a total obstruction of her left ilio-femoral vein with an extension to the vena cava. She was treated with heparin followed by oral anticoagulant therapy. The four other affected members (mother, aunts and uncle of the propositus) had also presented recurrent thrombosis with onset at a young age. The grandfather, not tested, had died from massive pulmonary embolism at age 54. The immunological assay of protein S was performed in plasma by Laurell, using a monospecific antiserum to human protein S, or by an ELISA, using a kit from Diagnostica Stago (Asserachrom Protein S). In order to separate free protein S, the functionally active form, from protein S complexed with C4-binding protein, plasma was adsorbed with 3.75 % polyethyleneglycol (PEG 6000). Following PEG precipitation, the levels of free protein S antigen remaining in the supernatant were quantitated by the usual immunological methods. In addition, two-dimensional immunoelectrophoresis (DDIE) also provided information on the distribution of both forms. In plasma protein S levels were decreased (40 to 55 % of the normal range) in two untreated patients and lower levels (17 to 20 96) were observed in the three others, including the propositus, who were under dicoumarol therapy. In PEG treated-plasma, protein S was undetectable (less than 5 %) in all patients, indicating a lack of free protein S. This was confirmed by DDIE : whereas protein S migrated as two distinct peaks, corresponding to free and complexed protein S in normal plasma, only a single peak of complexed protein S was observed in all affected patients. These results clearly demonstrate a total lack of free protein S which appears to be responsible for the thromboembolic disorder in this family as there was no deficiency of the other plasma inhibitors (antithrombin III, heparin cofactor II and protein C). According to the classification recently proposed by Comp et al., this family belongs to type I protein S deficiency, with an autosomal dominant mode of inheritance.