The in vivo synthesis of arginine from glutamate in mammals requires seven enzymes to cooperate. Pyrroline-5-carboxylate synthase (PCS) is the first enzyme required. In order to establish the interorgan dependency of arginine synthesis, we quantitated PCS activity in as many as 32 rat tissues and found that the activity was concentrated only in the upper small intestine. Minor activity was found in pancreas, thymus, lymph node, and some other tissues: this was confirmed by the dependency on specific substrates, the loss of activity in the presence of an inhibitor, and identifying the reduced product as proline. No difference in activity was found between male and female rats on a milligram protein basis. The strict tissue localization of PCS and the localization of other enzymes of arginine synthesis previously reported clearly indicate that the upper small intestine is an indispensable tissue for the arginine synthesis from glutamate. Many of the tissues examined showed an activity to form an unknown product from glutamate. When assayed by the previously reported radiometric assay procedure using an AG1-X8 column (acetate), the product was not separated from PC and caused false-positive activities of PCS. An improved procedure was developed to overcome this technical difficulty. The new procedure enabled us to detect even 20 pmol PC without contamination by the adjoining unknown product. A preliminary characterization of the unknown product was achieved.