The stereochemical course of the phospho transfer catalyzed by homogeneous human prostatic acid phosphatase was investigated using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from phenyl-(R)-[15O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This stereochemical result is consistent with the interpretation that the hydrolysis of substrates by this enzyme proceeds by way of a covalent phosphoenzyme intermediate. Conditions for optimizing phospho transfer by this and related acid phosphatases have also been explored.