The thrombin-catalyzed activation of single chain bovine Factor V was found to proceed by cleavage of the parent molecule (Mr = 330,000) to intermediates of apparent Mr = 205,000 and 150,000 followed by cleavage of the latter to end products of 94,000 and 74,000 apparent molecular weights. The generation of Factor Va activity coincides with the second cleavage. Further cleavages of the Mr = 205,000 component to apparent Mr = 74,000 and 32,000 fragments and possibly to others also occurred at a slower rate with little or no further increase in activity. The nature of the products and intermediates was unchanged regardless of whether the activation process was complete in either 1 or 10 min. Factor Xa did not catalyze the cleavage nor enhance the activity of Factor V under conditions in which the activation by comparable amounts of thrombin was complete. It was shown using either discrete clotting assays or continuous thrombin analysis with a chromogenic substrate that Factor V was subject to activation in situ by newly formed thrombin in a system initially containing prothrombin, Factor Xa, Ca2+, phospholipid, and unactivated Factor V. The rate of activation of prethrombin 1 was found to be saturable with added Factor Va when all other components were present at fixed levels. The amount of Factor Va required to achieve saturation was linearly dependent on the amount of Factor Xa present, indicating that the two factors interact stoichiometrically in the "prothrombinase" complex.