Alkaline phosphatase from mouse intestinal epithelial cells catalyzes the synthesis of pyrophosphate from Pi during hydrolysis of either glucose 6-phosphate, ATP, ADP, inorganic pyrophosphate or p-nitrophenylphosphate. The rate of pyrophosphate synthesis is increased by MgCl2 and by decreasing the pH of the medium from 8.5 to 6.0. The data presented indicate that at the catalytic site of alkaline phosphatase the energies of hydrolysis of the phosphoserine residue and of pyrophosphate are different from those measured in aqueous solutions.