Type III protein S deficiency is characterized by a low plasma level of free protein S, whereas the total concentration of protein S is normal. In contrast, both free and total protein S levels are low in type I deficiency. To elucidate the molecular mechanism behind the selective deficiency of free protein S in type III deficiency, the relationship between the plasma concentrations of beta-chain containing isoforms of C4b-binding protein (C4BP beta+) and different forms of protein S (free, bound, and total) was evaluated in 327 members of 18 protein S-deficient families. In normal relatives (n = 190), protein S correlated well with C4BP beta+, with free protein S (96 +/- 23 nmol/L) being equal to the molar excess of protein S (355 +/- 65 nmol/L) over C4BP beta+ (275 +/- 47 nmol/L). In protein S-deficient family members (n = 117), the equimolar relationship between protein S (215 +/- 50 nmol/L) and C4BP beta+ (228 +/- 51 nmol/L), together with the high affinity of the interaction, resulted in low levels of free protein S (16 +/- 10 nmol/L). Free protein S levels were distinctly low in protein S-deficient members, whereas in 47 of the protein S-deficient individuals, the concentration of total protein S was within the normal range, which fulfils the criteria for type III deficiency. The remaining 70 had low levels of both total and free protein S and, accordingly, would be type I deficient. Coexistence of type I and type III deficiency was found in 14 families, suggesting the two types of protein S deficiency to be phenotypic variants of the same genetic disease. Interestingly, not only protein S but also C4BP beta+ levels were decreased in orally anticoagulated controls and even more so in anticoagulated protein S-deficient members, suggesting that the concentration of C4BP beta+ is influenced by that of protein S. In conclusion, our results indicate that type I and type III deficiencies are phenotypic variants of the same genetic disease and that the low plasma concentrations of free protein S in both types are the result of an equimolar relationship between protein S and C4BP beta+.