C-14-labeled nicotinamide cofactors are widely employed in biomedical investigations: for example, to delineate C-14-labeled nicotinamide cofactors are widely employed in biomedical investigations, for example, to delineate metabolic pathways, to elucidate enzymatic mechanisms, and as substrates in kinetic isotope effect (KIE) experiments. The C-14 label has generally been located remote from the reactive position, frequently at the adenine ring. Rising costs of commercial precursors and disruptions in the availability of enzymes required for established syntheses have recently made the preparation of labeled nicotinamides such as [Ad-C-14]NADPH unviable. Here, we report the syntheses and characterization of several alternatives: [carbonyl-C-14]NADPH, 4R-[carbonyl-C-14, 4-H-2]NADPH, and [carbonyl-C-14, 4-H-2(2)]NADPH. The new procedures use [carbonyl-C-14]nicotinamide as starting material, because it is significantly cheaper than other commercial C-14 precursors of NADPH, and require only one commercially available enzyme to prepare NAD(P)(+) and NAD(P)H. The proximity of carbonyl-C-14 to the reactive center raises the risk of an inopportune C-14 isotope effect. This concern has been alleviated via competitive KIE measurements with Escherichia coli dihydrofolate reductase (EcDHFR) that use this specific carbonyl-C-14 NADPH. A combination of binding isotope effect and KIE measurements yielded no significant C-12/C-14 isotope effect at the amide carbonyl (KIE = 1.003 +/- 0.004). The reported procedure provides a high-yield, high-purity, and cost-effective alternative to labeled nicotinamide cofactors synthesized by previously published routes. (C) 2012 Elsevier Inc. All rights reserved.