We genetically screened patients with hereditary protein S deficiency for heterozygosity of protein S mRNA using PCR-RFLP for Pro626 polymorphism. All patients who showed hemizygous state of protein S mRNA, characterized by markedly decreased levels of one allele, had a phenotype of type I protein S deficiency. A putative mutation, such as a nonsense or splice site mutation, in the silent alleles may have affected the mutated mRNA metabolism and reduced the mutated mRNA accumulation, and consequently resulted in type I protein S deficiency in these patients. We also applied this mRNA-based analysis to family studies in hereditary protein S deficiency. In a family with type I protein S deficiency, all affected individuals showed a loss of one allele at the mRNA level and the silent allele cosegregated with the disease phenotype. Detection of hemizygous expression of protein S mRNA provided direct evidence for type I protein S deficiency without further precise genetic analysis. Our findings indicate that this mRNA-based analysis can be a useful strategy for genetic screening and family studies in hereditary protein S deficiency.