Gluconolactonase is isolated and purified from beef liver. The molecular weight is estimated at 233,000 and that of its six similar subunits is 39,400. The pH maximum is 7.1 in 50 mm Tris-acetate buffer at 27 °C. Km and Vm values of 9.1 mm and 1.62 mmol/min/ mg, respectively, were obtained at 27 °C in 50 mm Tris-HCl buffer. This enzyme requires a divalent metal for activity, with manganese being preferred over magnesium. A subcellular fractionation study indicates that gluconolactonase is located primarily in the cytosol, and its hepatic concentration is 2.3 ?mol/kg of hepatic tissue. © 1979.