The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.