The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino-naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.