Human protein S (HPS) is a vitamin K dependent plasma glycoprotein involved in the regulation of activated protein C and possibly fibrinolysis. Its c-DNA sequence shows three N-glycosylation consensus sequences (Asn-X-Ser/Thr). In order to study influence of N-linked glycosylation on HPS function, set of mutants of HPS was constructed. Mutants were generated, starting from an SV40/Adeno derived pD5HPS2 expression vector, using PCR enabled, site specific methodology. They included single amino acid substitutions at each of three N-glycosylation consensus sequences: Asn458-->Gln, Ser460-->Gly, Asn468-->Gln, Thr470-->Gly, Asn489-->Gln, Thr491-->Gly. Variant HPS were expressed in stable 293 human kidney cell lines in the presence of vitamin K1 (we did not succeed in expressing variant Asn489-->Gln) and purified from conditioned media using pseudoaffinity chromatography on QAE-Sepharose. Variant Asn468-->Gln showed decreased gamma-carboxyglutamate content. All of the mutants were active in a clotting type assay based on factor Va inactivation, and they were compared to wt-HPS and plasma HPS. In conclusion, we have constructed, expressed and purified set of HPS mutants useful in studying the role of N-glycosylation in HPS function.