To determine the molar absorptivities of β-NADH and β-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with εβ-NADH340 = 6.22 x 103 for β-NADH at 260 nm and 25 °C, a molar absorptivity of 14.1 x 103 liter · mol-1 · cm-1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of β-NAD at 260 nm and 25 °C was established to be 17.4 x 103 liter · mol-1 · cm-1. The values observed are lower than those reported thus far.