In the presence of phospholipid vesicles and calcium ions, protein Z (PZ) serves as a cofactor for the inhibition of coagulation factor Xa by a plasma protein called PZ-dependent protease inhibitor (ZPI). To further characterize ZPI, its cDNA has been isolated and cloned from a human liver cDNA library. The ZPI cDNA is 2.44 kb in length and has a relatively long 5' region (466 nt) that contains six potential ATG translation start codons. ATG's 1-4 are followed by short open reading frames, whereas ATG5 and ATG6 are in an uninterrupted open reading frame that includes the encoded ZPI protein. In vitro experiments show that ATG6 is sufficient for the expression of rZPI in cultured Chinese hamster ovary cells. Northern analysis suggests the liver is a major site of ZPI synthesis. The predicted 423 residue amino acid sequence of the mature ZPI protein is 25-35% homologous with members of the serpin superfamily of protease inhibitors and is 78% identical to the amino acid sequence predicted by a previously described cDNA isolated from rat liver, regeneration-associated serpin protein-1 (rasp-1). Thus, ZPI is likely the human homologue of rat rasp-1. Alignment of the amino acid sequence of ZPI with those of other serpins predicts that Y387 is the P1 residue at the reactive center of the ZPI molecule. Consistent with this notion, rZPI(Y387A), an altered form of ZPI in which tyrosine 387 has been changed to alanine, lacks PZ-dependent factor Xa inhibitory activity.