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To assess current laboratory practice and the performance of different reagent-instrument combinations for protein S testing, protein S results from the North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency testing surveys for 2002 and the first half of 2003 were analyzed. A written survey of NASCOLA laboratories also was performed to further assess current laboratory practices for protein S testing. The free protein S antigen assays and the Diagnostica Stago Staclot protein S assay were extremely accurate in detecting a heterozygous type I protein S deficiency. Another functional protein S assay and most total protein S assays were less reliable, depending to some extent on the instrument. All assays used by NASCOLA laboratories appropriately identified normal protein S specimens. NASCOLA laboratories performed at least as well as European Concerted Action on Thrombosis laboratories in the proficiency tests. The results suggest that the diagnosis of heterozygous protein S deficiency may be problematic with some currently available assays. Many total protein S antigen assays do not add to the diagnosis and can be unreliable for protein S deficiency subtyping. Better standardization of functional and antigenic assays is needed.