The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.