SYNOPSIS A very rapid method of agar gel electrophoresis on glass slides, together with a superior visualization technique employing simultaneous coupling of a hydrolysed naphthol substrate, have been developed for the identification of the tissues of origin of serum alkaline phosphatase. Combined with L-phenylalanine inhibition, specific for the intestinal enzyme, and heat inactivation, specific for the placental enzyme, the heterogeneity of serum alkaline phosphatase has been demonstrated. Normal adult serum contains predominantly liver-type alkaline phosphatase with a small but variable quantity of intestinal enzyme, and little or no bone enzyme.In childhood and in infancy there is in addition a bone isoenzyme present, the amount gradually falling to adult levels with age. In pregnancy, the rise in serum alkaline phosphatase is due to the placental enzyme.A study of nearly 2,000 sera has been undertaken and it is found that the bone enzyme is increased in osteoblastic bone diseases while in hepato-biliary disorders there is an increase in liver type enzyme. The main theories explaining the rise in serum alkaline phosphatase are examined.The origin of serum alkaline phosphatase has interested many workers ever since Kay (1929) and Roberts (1930) first demonstrated that increased serum activity was found in certain bone and hepatobiliary disorders. The problem of the origin of the enzyme is intricately bound up with that of the mechanisms by which the serum activity is increased or decreased in health and in disease. It has long been realized that serum alkaline phosphatase arises from tissues rich in the enzyme (Kay, 1932). Previous attempts at identification of the tissues of origin were handicapped not only by lack of direct methods, but also by the influence of the work of Robison and his associates linking alkaline phosphatase with calcification (