A cDNA coding for the carboxy-terminal region of human protein S and containing a complete 3'-untranslated region, was isolated by a combination of antibody screening of a lambda gt11 human liver cDNA expression library and in situ hybridization of a pUC9 human liver cDNA library. Hybridization analysis of human total DNA with radiolabelled non-overlapping cDNA restriction fragments revealed the existence of two genes per haploid genome homologous with the protein S cDNA. Both genes were mapped to chromosome 3 using human-rodent cell hybrids. Neither of the genes showed polymorphism for sixteen different enzymes upon hybridization with the protein S cDNA.