The kinetic parameters of the conversion of bovine prothrombin into thrombin by activated bovine blood clotting factor X (Xa) have been determined in the absence and presence of Ca2+, activated bovine factor V (Va) and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 1:1; mol/mol). In the absence of accessory components, the Km for prothrombin is 131 microM, which is well above its concentration in bovine plasma of about 1.5 microM. The Vmax of thrombin formation is 0.61 mol min-1 mol of Xa-1 under these conditions. In the presence of 7.5 microM phospholipid, the Km drops to 0.058 microM and the Vmax slightly increases to 2.25 mol min-1 mol of Xa. For the complete prothrombinase complex (Xa, Va, Ca2+, and 7.5 microM phospholipid), a Km for prothrombin of 0.21 microM and a Vmax of 1919 mol min-1 mol of Xa-1 is found. The Vmax of thrombin formation slightly increases when more phospholipid is present in our experiments and there is a considerable increase of the Km for prothrombin at higher phospholipid concentrations. Preliminary calculations show that the prothrombin density at the phospholipid surface at the Km is independent of the phospholipid concentration. This indicates that the Km measured in the presence of phospholipid has to be regarded as an apparent Km and the local prothrombin concentration determines the kinetics of activation. Prothrombin activation by prothrombinase complexes of different compositions was followed by gel electrophoresis in the presence of sodium dodecyl sulfate. Both in the absence and presence of phospholipid but without factor Va, prethrombin 2 is the main product formed during the initial stages of steady state prothrombin activation. In the presence of factor Va, thrombin is the main end product and minute amounts of prethrombin 2 are formed. This shift in the reaction pathway of prothrombin activation caused by factor Va will contribute to the observed increase of the Vmax measured in the presence of factor Va.