A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NH units/mg of protein , a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroesis, E 1.0 mg/ml 1.0 cm, 280 nm = 1.45 at pH 7.0 and the following amino acid composition: Asx 51, Thr 38, Glx 62, Pro 23, Gly44, Ala 25, Half-Cys 30, Val 35, Met 3, Ile 30, Leu 32, Tyr 19, Phe 22, Lys 36, His 8, Arg 28, and Trp 13, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine was found as the only N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.