Abstract The alkaline phosphatases present in the human tissues liver, kidney, intestine, placenta and a serum from a patient with Paget's disease of bone have been purified to apparent homogeneity by affinity elution from a phosphonic acid-Sepharose derivative. Polyacrylamide gel electrophoresis in SDS 1 gave sub-unit molecular weights ranging from 74 000 for the enzyme from placenta to 95 800 for the enzyme from kidney. The purified native and desialylated enzymes have been characterized by agar electrophoresis and isoelectric focusing. All five of the native enzymes behaved differently but the desialylated forms from liver, Paget's serum and kidney were indistinguishable in both systems. The desialylated enzyme from placenta and the enzyme from intestine behaved differently from each other and from the above mentioned group. The isoelectric points ranged from < 4 for the native enzyme from liver to 7.01 for the desialylated forms of the enzymes from liver, kidney and Paget's serum. The effects of a number of factors on the thermostability of the purified enzymes were studied. Phosphate decreased the stability and human serum albumin increased the stability of most molecular forms. Desialylation had no effect on thermostability. These results and those from inhibition studies with l-phenylalanine, l-homoarginine, phosphate and vanadate support the 3-gene hypothesis advanced from structural studies.