The reduction potential of the (FADH-/FADH•) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 ± 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 ± 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.