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SummaryA modified and improved method for isolation of calf intestinal alkaline phosphatase is described. By this method 300 to 400 mg of pure enzyme was prepared in a relatively short time. On the basis of the results of ultracentrifugation and of the free, polyacrylamide and immunoelectrophoresis the phosphatase obtained is found to be a homogenous glycoprotein, containing firmly bound zinc, magnesium and phosphoric acid. The molar composition of the enzyme and the catalytic activity were determined with different substrates and buffers.1. Introduction. -The affinity chromatography has been already introduced as an isolation procedure of the alkaline phosphatase of calf and human intestinal mucosa [ 1-41. This method certainly simplifies the rapid preparation of very small amounts of highly purified phosphatase, but the poor capacity of the ligandcharged resins does not allow an easy isolation of larger quantities of pure enzyme. Furthermore, there is no sufficient experimental proof, that the phosphatase preparations isolated by affinity chromatography are homogenous proteins. It is even doubtful, that the nature of the employed ligands, all having an ion-exchange sideeffect, and their rather low inhibitor specificity permit the isolation of a homogenous phosphatase. Theoretically none of these ligands can distinguish between the always present isoenzymes and phosphatase molecular variants easily produced during the solubilization of the membrane-bound enzyme. These enzyme variants have different sugar content and can, according to our experience, only be separated by the classical isolation methods. Our method, refined and tested during many years, opens a possibility with reproducibility.