Lactase preparations were purified from cell-free extracts of Kluyveromyces lactis to homogeneity, as determined by disc SDS polyacrylamide gel electrophoresis. A combination of techniques including ammonium sulfate or acetone precipitation and hydroxyapatite chromatography was used for the purification of the enzyme. The enzyme has a pH optimum of 6.5-7.0 with Kms' of 1.25 and 28mM for the substrates O-nitrophenylgalactopyranoside and lactose respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 15.1 and 10.0 Kcal/mol, respectively. When reconstituted skim milk containing 20% total solids was treated with the lactase preparation (60,000 lactase units/1 of milk) at 18°C, 82% of the milk lactose was hydrolyzed within 24 h. Such treatment lends itself for application in developing countries and where the incidence of lactose intolerance is high.