A protein inhibitor to ornithine decarboxylase, antizyme, was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 600,000-fold with a 15% yield from the liver cytosol of putrescine-treated rats. Antizyme was very labile but markedly stabilized in the presence of Tween 80 and 2-mercaptoethanol. The apparent molecular weight of antizyme was determined to be 22,000 by gel filtration on Sephadex G-75 and 19,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that antizyme may be composed of a single polypeptide chain. The isoelectric point of antizyme was found to be 6.8. The equilibrium constant of the reaction between antizyme and ornithine decarboxylase was estimated to be as high as 1.4 X 10(11) M-1. Some other properties of antizyme were also described.