We herein report that a novel membrane-associated protease capable of activating prothrombin is present in several mammalian cells. This protease can directly convert prothrombin to active thrombin and induces blood clotting both in vivo and in vitro but is apparently different from coagulation factor Xa, which has been thought to be the only physiological activator of prothrombin. This protease activity was initially found and was very high in 8C feline kidney fibroblast cells, and we characterized its enzymological features using this cell line. Activity was detected in neither the cytosolic fraction nor the culture medium but found in the membranes and identified on the surface of intact cells. The activation of prothrombin required Ca2+ ions, and the apparent Km value for prothrombin was 0.2 microM. The activity was irreversibly inhibited by exposure to EDTA, but various inhibitors for serine proteases including antithrombin III were without effect. Based on these results, we propose that this novel enzyme, membrane-associated prothrombin activator, catalyzes an alternative pathway for generation of thrombin, which is independent of the blood coagulation cascade, and that the thrombin generated is involved in certain pathological states and/or in activation of cells that are spatially separated from the bloodstream.