Haemophilia B is characterized by a deficiency of the gamma-carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach (Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K-dependent proteins in vitro, to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT-FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is gamma-carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.