Prothrombin has been purified from rat plasma and its properties compared to prothrombin isolated from other species. The molecular weight, amino acid composition, and amino-terminal sequence of rat prothrombin are similar to human and bovine prothrombin. Rat prothrombin binds to phospholipid in the presence of calcium ions, and calcium-binding measurements indicate that it may bind somewhat more calcium than does bovine prothrombin. The proteolytic cleavage of purified rat prothrombin by Factor Xa or thrombin yields the same peptides that are formed from similar proteolysis of bovine prothrombin. Factor V and phospholipid were shown to enhance the rate of Factor Xa and calcium ion generation of thrombin from rat prothrombin.