Antithrombin III was purified from rat plasma in 70% yield by salting out with (NH4)2SO4, affinity chromatography on heparin-Sepharose 4B, and ion-exchange chromatography on DE-52. The preparation was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, by analytical ultracentrifugation, and by immuno-chemical analysis. It was composed of a single polypeptide chain whose molecular weight was estimated to be about 64 000 both by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by sedimentation equilibrium analysis. Antithrombin III was a glycoprotein containing 3.6% glucosamine, 0.2% fucose, 2.5% mannose, 1.6% galactose and 3.9% sialic acid. Isoelectric focusing in polyacrylamide gel revealed four bands in the range of pH 4.7--4.9, indicating the microheterogeneity. Rat antithrombin III inhibited bovine alpha-thrombin by forming an equimolar complex. Kinetics of this reaction were studied by gel electrophoresis in sodium dodecyl sulfate. When the inhibitor was allowed to react with an excess amount of thrombin, the initial equimolar complex with a molecular weight of 110 000 was cleaved gradually to a product with a molecular weight of 97 000, which was further cleaved to a second product with a molecular weight of 85 000.