A cDNA library prepared from human liver has been screened for factor IX (Christmas factor), a clotting factor that participates in the middle phase of blood coagulation. The library was screened with a single-stranded DNA prepared from enriched mRNA for baboon factor IX and a synthetic oligonucleotide mixture. A plasmid was identified that contained a cDNA insert of 1,466 base pairs coding for human factor IX. The insert is flanked by G-C tails of 11 and 18 base pairs at the 5' and 3' ends, respectively. It also included 138 base pairs that code for an amino-terminal leader sequence, 1,248 base pairs that code for the mature protein, a stop codon, and 48 base pairs of noncoding sequence at the 3' end. The leader sequence contains 46 amino acid residues, and it is proposed that this sequence includes both a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 1,248 base pairs code for a polypeptide chain composed of 416 amino acids. The amino-terminal region for this protein contains 12 glutamic acid residues that are converted to gamma-carboxyglutamic acid in the mature protein. These glutamic acid residues are coded for by both GAA and GAG. The arginyl peptide bonds that are cleaved in the conversion of human factor IX to factor IXa by factor XIa were identified as Arg145-Ala146 and Arg180-Val181. The cleavage of these two internal peptide bonds results in the formation of an activation peptide (35 amino acids) and factor IXa, a serine protease composed of a light chain (145 amino acids) and a heavy chain (236 amino acids), and these two chains are held together by a disulfide bond(s). The active site residues including histidine, aspartate, and serine are located in the heavy chain at positions 221, 270, and 366, respectively. These amino acids are homologous with His57, Asp102, and Ser195 in the active site of chymotrypsin. Two potential carbohydrate binding sites (Asn-X-Thr) were identified in the activation peptide, and these were located at Asn157 and Asn167. The homology in the amino acid sequence between human and bovine factor IX was found to be 83%.