1. The enzymatic nature of human liver, bone, placental and intestinal alkaline phosphatases (ALPs) were investigated with phosphorylcholine (PC), phosphorylethanolamine, pyridoxal-5'-phosphate and p-nitrophenylphosphate at a weakly alkaline pH. 2. The apparent Km value of the intestinal ALP with PC was the highest of all ALPs tested. Intestinal ALP hydrolyzes PC the most and has higher affinity for choline as a transphosphorylating acceptor than the other ALPs. In addition, the intestinal ALP activity with PC was most susceptible to Na2HPO4, in the tested ALPs. 3. The present results suggest that PC is a unique substrate for human intestinal ALP, which may be related to the metabolism of PC or choline as part of phosphatidylcholine.