The rates of leucine aminopeptidase hydrolysis of O-phosphorylated, O-monophenylphosphorylated, and O-pyrophos-phorylated serine peptides were determined and compared with those of the corresponding phosphate-free peptides. A phosphate group near the free [alpha]-amino end, as in N-terminal O-phosphorylserine peptides, lowers the rate of hydrolysis more than 100 times, and a pyrophosphate group in the same position more than 1000 times. O-Monophenylphosphorylated peptides are hydrolyzed at about the same rate as their phosphate-free analogues.