Factor V must be converted to Factor V(a) in order to bind to a high affinity platelet surface site and participate in prothrombin activation. Osterud et al. (10) presented data that suggested that human platelets contain an activated form of Factor V and a Factor V activator. We find that the Factor V released when platelets are disrupted by freezing and thawing or sonication is activated 3- to 10-fold by thrombin as determined by coagulation assay and is therefore stored as the relatively inactive procofactor rather than in the active form Factor V(a). We incubated purified Factor V, which had a specific activity of 140+/-30 U/mg, with Factor V-deficient frozen and thawed platelets (10(9) platelets/ml) obtained from a patient with Factor V deficiency. The specific activity of the Factor V increased to a maximum of 740+/-240 U/mg (mean+/-SD of three experiments). When this partially activated Factor V was incubated with thrombin its specific activity increased further to 1,440+/-280 U/mg, which is similar to the activity of Factor V activated with thrombin alone (1,540+/-60 U/mg). The platelet Factor V activator is not inhibited by dansyl arginine-4-ethylpiperidine amide, 93 muM, indicating that it is not thrombin. When thrombin-stimulated platelets, to which dansyl arginine-4-ethylpiperidine amide had been added to inhibit the further action of thrombin, were incubated with (125)-labeled Factor V, there was no detectable proteolysis of the Factor V molecule. Our failure to detect activation of Factor V under these conditions suggests that <4% of the platelet protease is released by thrombin. Subcellular fractionation of platelets indicates that the platelet protease that activates Factor V is in the soluble fraction. When Factor V(a) formed by the action of platelet protease is incubated with platelets, peptides with M(r) = 105,000, 87,000, and 78,000 bind to the platelet surface. All three radiolabeled peptides are displaced from platelets by unlabeled Factor V(a) formed by the action of thrombin. The stoichiometry of binding suggests that the 105,000-dalton peptide is associated with either an 87,000- or a 78,000-dalton peptide. The 78,000-dalton peptide binds with greater affinity and probably accounts for the bulk of the activity of Factor V(a) in coagulation assays. Whether or not the platelet protease serves to activate Factor V before thrombin formation during normal hemostasis remains to be determined.