A model system for the analysis of intracellular events governing the modification of individual vitamin K-dependent (VKD) proteins by the carboxylase has been developed using recombinant VKD protein-transfected cell lines. When untransfected 293 cells were analyzed by in vitro carboxylation followed by SDS-PAGE, endogenous VKD proteins were not detected. With 293 cells stably-transfected with recombinant native factor IX, most (> 95%) of the carboxylase was in complex with the factor IX, as assayed by adsorption of carboxylase activity to immobilized anti-factor IX antibody. In contrast, with 293 cells stably-transfected with recombinant factor IX deleted in the propeptide sequence (amino acids -18 to -4, delta pro factor IX), no association of factor IX with the carboxylase was observed. This observation was used to specifically isolate and identify the human carboxylase, and carboxylase-associated protein. When the carboxylase was purified from solubilized microsomes from either native factor IX, or delta pro factor IX, stably-transfected 293 cells, a single 98 kDa band was specifically obtained from native factor IX microsomes, but not from delta pro factor IX microsomes. This band was subsequently shown by Western and microsequencing analysis to comprise both the carboxylase and carboxylase-associated protein. This isolation, which represents the first isolation to near homogeneity of both the human carboxylase and the carboxylase from cell lines, will be valuable in isolating enzymatically active recombinant carboxylase, which has been refractile to other purification attempts. This system was also used to show that the human carboxylase in 293 cells is capable of binding and modifying two different liver-derived proteins. Protein C-producing 293 cells were generated from the same 293 progenitor cell line used to created the factor IX-expressing cells. With both factor IX- and protein C-transfected 293 cells, the secreted proteins were almost completely carboxylated, and in microsomes from each cell line the carboxylase was found in near quantitative complex with the two different VKD proteins. Thus the carboxylase modifies both VKD proteins. The approach described here for the analysis of the carboxylase from recombinant VKD protein-transfected cell lines should provide an important new system for studying protein carboxylation and VKD protein-carboxylase interaction.