Abstract A simplified procedure is described for the large-scale purification of bovine prothrombin complex. The protein was adsorbed on barium carbonate, eluted with 3.5% sodium citrate solution, and fractionated with ammonium sulfate. Additionally, prothrombin was separated from autoprothrombin III (Factor X) and further purified by DEAE-cellulose chromatography. Prothrombin in the latter preparation was then obtained in pure form by two sequential filtrations through a Sephadex G-100 column. The specific activity was 3800 u/mg protein. The N-terminal amino acid was alanine. By ultracentrifugation, a single component was indicated. The equation for S 20,w o was 4.75 + 0.0196X. Eight mg% is a revised figure for the concentration of prothrombin in normal bovine plasma.