Factor IX and its activated form IX(a) have been found to bind to confluent cultured bovine aortic and human umbilical vein endothelial cells. Binding of bovine factors IX and IX(a) to the bovine endothelial cells was saturable and specific and reached a plateau in 75 min at 4°C and 30 min at 37°C. Binding was half-maximal at a total factor IX or IX(a) concentration of 2.3 ± 0.2 nM. At 4°C, a maximum of 42 fmol of tritiated factor IX or IX(a) bound to 106 cells (an average of 20,000 molecules per cell). The binding of tritiated factor IX or IX(a) was inhibited by excess unlabeled factor IX or IX(a) but not by factor X, prothrombin, or thrombin. Competition studies indicated that factors IX and IX(a) interacted with the same site. Binding was reversible, with 50% of the specifically bound factor IX or IX(a) eluted in 40 min by a 400-fold excess of unlabeled protein. Specific binding required Ca2+ with half-maximal binding at 1.2 mM CaCl2. Factor IX(a) bound to the cells was tested for procoagulant activity in a clotting assay with factor IX-deficient plasma, cephalin, and CaCl2. Cell-bound factor IX(a) was at least 3-fold more active than was factor IX(a) in solution. The retention of procoagulant activity by cell surface-bound factor IX(a) provides a mechanism for the localization of clot-promoting activity.