We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTMα (low salt elution) and rsTMβ (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTMβ contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of α and β were 7.4 and 1.4 nM respectively. RsTMβ was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTMα. Both anticoagulant activities were lost after chondroitinase treatment of rsTMβ.