[14C]Pyridoxal 5′-phosphate was synthesized by first oxidizing [14C]pyridoxine to [14C]pyridoxal with MnO2. [14C]Pyridoxal, isolated by cation-exchange chromatography, was then reacted with ATP in the presence of rat liver pyridoxal kinase to form [14C]pyridoxal 5′-phosphate. The rat liver enzyme was used instead of the Escherichia coli pyridoxal kinase because the latter is strongly inhibited by pyridoxal. [14C]Pyridoxal 5′-phosphate was purified by Sephadex G-10 chromatography. The yield of the present method is 75%. The radiochemical purity of [14C]pyridoxal 5′-phosphate produced is ≥95%. © 1981.