Uniformly labeled (U) 14C nicotinamide adenine dinucleotide phosphate (NADP+) was synthesized by phosphorylating [U-14C]nicotinamide adenine dinucleotide (NAD+) in the presence of immobilized NAD+ kinase. The 15 μCi (600 μL) synthesis consistently achieved yields between 80% and radiochemical purities greater than 95%. The [U-14C]NADP+ was purified by high performance anion-exchange chromatography using a gradient elution of ammonium bicarbonate. This procedure may be applicable to the synthesis of other charged, UV-absorbing products of enzyme-catalyzed reactions.