Protein S is an anticoagulant protein that circulates in plasma in complex with C4b-binding protein (C4BP) or in free form. Deficiency of protein S increases the risk of venous thrombosis. Measurement of free protein S, as compared to total levels, has been shown to be superior for prediction of protein S deficiency. We studied the effects of different handling protocols for an immuno- and a ligand (C4BP)-based assay for free protein S. When the assay was performed at 37 degrees C, the levels of free protein S in plasma from protein S deficient patients were approximately twice those obtained at room temperature. The reason for this phenomenon was that plasmas from protein S deficient patients exhibited a time-, temperature-, and dilution-dependent increase in free protein S, which was more pronounced than corresponding dilution of the normal plasma that was used to create the standard curve. These findings demonstrate the importance of assay procedure and sample handling in assays for free protein S.