The three phospholipase A2 isoenzymes from Naja nigricollis venom inhibit blood coagulation with different potencies. The strongly anticoagulant basic isoenzyme CM-IV inhibits the prothrombinase complex, whereas the weakly anticoagulant isoenzymes CM-I and CM-II do not. To determine the role of enzymatic activity of the phospholipases in the inhibition of prothrombinase, we varied the time of incubation of each of these isoenzymes with the prothrombinase complex. The inhibition by CM-IV did not increase with time of incubation. CM-I and CM-II failed to inhibit the complex, even with complete hydrolysis of phospholipids in the assay mixture. After alkylation of its active-site histidine, CM-IV lost 97% of its enzymatic activity but retained 60% of its inhibitory potency on prothrombinase. CM-IV also inhibited prothrombinase activity in the absence of phospholipids, whereas CM-I and CM-II did not. The inhibition of the prothrombinase complex by CM-IV is thus not due to its binding to or hydrolysis of phospholipids. The kinetics of CM-IV inhibition of the prothrombinase complex in both the presence and absence of phospholipids was noncompetitive. This inhibition can be explained by binding of CM-IV to either factor Va or Xa, or both, to inhibit the complex. CM-IV differs from previously described nonenzymatic anticoagulants that are proteinase inhibitors or that inhibit the coagulation complexes by interfering with the binding of clotting factors to phospholipids. We conclude that the basic enzyme, CM-IV, inhibits the prothrombinase complex by a novel mechanism independent of enzymatic activity.