Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeneity from the livers of thioacetamide- and DL-alpha-hydrazino-delta aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5.10(5)-fold with an 8% yield; the specific activity of the final enzyme preparation was 1.1.10(6) nmol CO2/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50000 on SDS-polyacrylamide gels. The isoelectric point of the enzyme was pH 4.1.