The enzyme which catalyzes the conversion of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate has been purified to homogeneity from a crude extract of beef liver. Analysis of the purified enzyme by Sephadex G-100 gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed it to be a dimer of relative molecular mass 43000. From the gas chromatography/mass spectrometry analyses of the enzymatic reaction products, it appeared that about 90% of the total amount of tetrose 4-phosphate was present as D-erythrulose 4-phosphate after equilibration. The purified enzyme, which is tentatively called 'erythrose-4-phosphate isomerase' had no significant isomerase activities on D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate, D-glucose 6-phosphate and D-fructose 6-phosphate, but a strong ~-ribulose-5-phosphate 3-epimerase activity was co-purified with the erythrose-4-phosphate isomerase activity through every step in the isolation. Both the erythrose-4-phosphate isomerase and ~-ribulose-5-phosphate 3-epimerase activities were inactivated at the same rate at the elevated temperature, and also inhibited to the same extent by various inhibitors. It is likey, that both activities are catalyzed by the single enzyme protein.We have reported previously the purification and properties of D-erythrulose reductase (EC 1.1.1.1 62) [I, 21 and aldehyde reductase (EC 1.1.1.2) [3], which catalyze the reduction of D-erythrulose and D-erythrose respectively. These enzymes were considered to be important with respect to the tetrose metabolism. In the continuing efforts from this laboratory to understand how the naturally occurring tetritols, such as erythritol and threitol excreted in urine, are synthesized we recently found an enzyme catalyzing both isomerization and epimerization of D-erythrose 4-phosphate (Ery4P) to D-erythrulose 4-phosphate and D-threose 4-phosphate in beef liver [4]. This enzyme could be described as erythrose-4-phosphate (Ery4P) isomerase since it was able to catalyze the isomerization of Ery4P to the major product, D-erythrulose 4-phosphate. Since other mammalian enzymes catalyzing such a reaction have not been reported until now, this enzyme has been the subject of extensive research by our laboratory.In the present report we describe the characterization of a highly purified enzyme having both the Ery4P isomerase and epimerase activities from beef liver, and discuss the mechanism