Although heparin acts as an anticoagulant by catalyzing the inactivation of thrombin (Ha) and factor Xa (Xa) by antithrombin (AT), heparin has little effect on the prothrombin time (FT). To explore this phenomenon, we examined the effect of several commercial thromboplastin reagents that do not contain heparin neutralizing agents on the heparincatalyzed second-order rate constants for lia and Xa inactivation by AT. The thromboplastin reagents, including one containing recombinant tissue factor (r-TF), reduced the heparin-catalyzed rates of Ha and Xa inhibition by approximately 40- and 60-fold, respectively. The reagent containing r-TF consists of r-TF relipidated with phospholipid micelles comprised of 2 mg phosphatidylcholine (PC), 0.5 mg phosphatidylserine (PS), 0.3 mg phosphatidylethanolamine (PE), and 0.2 mg phosphatidylglycine (PG) per ml. Although micromolar concentrations of r-TF alone produced a 10-fold reduction in the heparin-catalyzed rate of Ha and Xa inhibition, r-TF has no effect on these reactions when added in concentrations equivalent to those in the commercial thromboplastins. In contrast, with or without r-TF, phospholipid micelles comprised of either PCPSPEPG or PCPS produced the same reduction in the heparin-catalyzed rates of Ha and Xa inhibition by AT as did the thromboplastin reagents. When sonicated PCPSPEPG or PCPS vesicles at the same lipid concentrations were used in place of micelles, no effect on the rates was detected in the absence or presence of r-TF. The differences in activity between micelles and vesicles is not explained by the detergent used for micelle preparation because, on its own, the detergent produces only a 3-fold reduction in the heparin-catalyzed rate of Ha or Xa inhibition by AT. To determine whether PCPSPEPG micelles have the same activity in plasma as they do in buffer systems, the PT was measured in the absence or presence of heparin using r-TF relipidated with PCPSPEPG micelles. No prolongation of the PT was detected in the presence of up to 0.9 U/ml heparin. Similar results were obtained with the commercial thromboplastin reagents. In contrast, when r-TF lipidated with PCPSPEPG vesicles was used for PT determination, heparin produced a concentration-dependent prolongation of the clotting time such that the PT ratio was increased 10-fold with 0.9 U/ ml heparin. These data indicate that phospholipid micelles, but not vesicles, neutralize heparin, thereby explaining why heparin has minimal effects on the PT.