A survey of the hydrolytic activity of alkaline phosphatase (EC 3.1.3.1) reveals that PP(1), like phosphomonoesters, can serve as substrate in vitro. This PP(1) phosphohydrolytic activity can be distinguished from PP,-phosphohydrolytic activities of inorganic pyrophosphatases (EC 3.6.1,1) and glucose-6-phosphatase (EC 3.1.3.9) by several criteria. Discrimination among these hydrolytic enzymes is possible by their dependence on variation of pH and of magnesium to PP(1) ratios in the assay solutions. The true substrates and modifiers are not simply PP(1) and magnesium, but the equilibrium species in mixtures of these two. The physiological significance of each of the three enzymes is not predictable from their differential efficiency as catalysts of PP(1)-hydrolysis in vitro.