Factor IX is synthesized in a precursor form with a propeptide that contains the gamma-carboxylation recognition site, an element which directs the post-translational gamma-carboxylation of adjacent glutamic acid residues. After protein synthesis, the propeptide is cleaved to yield the mature Factor IX. To study propeptide processing, anti-proFactor IX antibodies were prepared using a synthetic peptide based upon the sequence of the Factor IX propeptide. Immunoaffinity-purified anti-proFactor IX antibodies were reactive with Factor IX Cambridge, a mutant form of Factor IX containing the propeptide, but were not reactive with Factor IX. These antibodies were used to examine the proteolytic processing of forms of Factor IX containing point mutations at P6, P3, P2, P1, P1', P2', and P3' adjacent to the propeptide cleavage site in order to determine the requirement of each of these amino acids for propeptide cleavage. Furthermore, the hierarchy of different pairs of basic residues at positions P1 and P2 was analyzed. The mutated cDNA constructs were expressed in Chinese hamster ovary cells. Propeptide processing was examined using intrinsically labeled Factor IX immunoprecipitated with either anti-pro-Factor IX antibodies or anti-Factor IX:total antibodies, and the Factor IX species were separated by SDS-gel electrophoresis. Under the expression conditions employed, the propeptide of wild type Factor IX was almost completely removed, whereas Factor IX mutated to threonine at P1 was not cleaved. The percentage of propeptide cleaved varied with the amino acid sequences of residues P2 and P1, respectively: Lys-Arg (93%), Arg-Arg (66%), Thr-Arg (33%), Arg-Lys (19%), Lys-Lys (10%), and Lys-Thr (< 1%). Apart from alterations of basic amino acids at P1 and P2, nonconservative mutations at P6 and P3 decreased propeptide cleavage, whereas conservative mutations at P3, P1', P2', or P3' resulted in cleavage efficiencies approximately equal to that for wild type Factor IX. These results indicate that the preference of paired basic residues at P1 and P2 is similar to other endopeptidases active toward proteins secreted through the constitutive pathway and that the propeptide residues NH2-terminal to these paired basic residues are important in defining enzyme-substrate binding.