This chapter describes the assay method, purification procedure, and properties of glucose-6-phosphate dehydrogenase from human erythrocytes. The activity of glucose-6-phosphate dehydrogenase is measured in the presence of excess glucose 6-phosphate and NADP by the increase in absorbance at 340 nm, which corresponds to the formation of NADPH. The reaction goes to completion since the product 6-phosphogluconolactone is rapidly hydrolyzed to 6-phosphogluconate. This hydrolysis is not a rate-limiting step in the standard assay. The maximum reaction velocity and Km are dependent on the ionic strength of the buffer, and are influenced by the particular ions present. For the purification procedure, blood is stored at 0–4° in acid-citrate-dextrose to prevent clotting, and the cells are centrifuged after each wash at 3000 g for 15 rain, and the supernatant is sucked off. A summary of the purification is also tabulated. On correction, the freshly prepared enzyme has a specific activity of 220 units/mg. This value declines to 150 nnits/mg over a period of 2 weeks. The preparation is homogeneous by the criteria of electrophoresis on starch gel at pH 8.6, 5 and on cellulose acetate at pH 7.5, and on polyacrylamide gels in the presence of sodium dodecyl sulfate. Some general properties of the enzyme and of the amino acid composition are summarized.