A partial cDNA clone for human antithrombin III (ATIII) was obtained by screening a cDNA library prepared from size fractionated liver RNA with a pool of eight 16-base long synthetic DNA fragments whose sequence was determined from protein sequence data. A fragment of the partial cDNA clone was used to enrich RNA for ATIII messages, and cDNA clones encoding the entire ATIII structural gene were identified. The complete nucleotide and predicted amino acid sequences of human ATIII and its 32 residue signal peptide are reported, and provide further opportunity to compare the ATIII primary structure with corresponding regions from homologous proteins, alpha 1-antitrypsin and ovalbumin. Plasmids in which the structural genes for mature and pre-ATIII were linked to the E. coli trp promoter-operator support the synthesis of human antithrombin III and pre-antithrombin III in bacteria.