A pure, stable preparation of chorismate mutase-prephenate dehydrogenase (chorismate pyruvatemutase, EC 5.4.99.5-prephenate:NAD+ oxidoreductase (decarboxylating), EC 1.3.1.12) has been obtained in good yield from a regulatory mutant of Escherichia coli. The enzyme was purified from extracts of the organism by treatment with streptomycin sulfate and fractionation with ammonium sulfate followed by chromatography on columns of Sepharose-AMP, DEAE-cellulose and hydroxyapatite. The native enzyme has a molecular weight of 88,000 and is made up of two identical subunits as indicated by the results of amino acid composition, peptide mapping and electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 4.85 S as determined in the ultracentrifuge and an isoelectric point of pH 5.3. Preliminary studies on the kinetic properties of the enzyme indicated that both the mutase and the dehydrogenase reactions catalyzed by the enzyme conform to Michaelis-Menten kinetics.