We have used a monoclonal antibody-based ELISA for plasma prothrombin fragment 1.2 (F1.2) to establish appropriate sample collection and storage conditions for this biomarker of thrombin generation. F1.2 concentrations were not altered by exogenous factor Xa, thrombin, or thromboplastin if blood was collected by routine venipuncture into tubes containing heparin as anticoagulant (but not citrate, acid-citrate-dextrose, EDTA, or oxalate) and if plasma antithrombin III concentration was > or = 30% of normal. Heparinized plasma F1.2 was stable for > or = 8 h at 20-25 degrees C, and if premixed with a stabilizing reagent, for > or = 4 years at -70 degrees C. Mean values for heparinized plasma F1.2 collected and stored by recommended procedures were increased in patients with thrombosis and conditions of increased thrombotic risk, and were sensitive to heparin and oral anticoagulant therapies. We conclude that plasma obtained by routine venipuncture into tubes with heparin as anticoagulant is an appropriate specimen for F1.2 measurements for most patients.