6-Phosphogluconolactonase was purified to apparent homogeneity in a four-step procedure from bovine erythrocytes. The extent of purification and the kinetic properties of the enzyme were evaluated with an optical test that was based on the hydrolysis of synthetic 6-phosphogluconolactone. The active enzyme from bovine erythrocytes is a monomer with an approximate molecular weight of 30000. It exhibits Michaelis-Menten kinetics and cofactors are not required for activity. The enzyme was found in a number of tissues. Its activity, when compared to the activity of the glycolytic enzymes, illustrates the importance of glucose oxidation via the pentose phosphate pathway relative to glycolysis.