Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus E1A. The finding that CtBP–E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD⁺ and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD⁺ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD⁺. The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD⁺ affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD⁺. Our studies show a >100-fold higher affinity of NADH than NAD⁺, consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD⁺ required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.