(\alpha)-NADH can be determined in the presence of high concentrations of (\beta)-NADH using anion exchange HPLC combined with oxidation of (\beta)-NADH by lactate dehydrogenase. The method is suited for the detection of a large number of impurities. Residual absorption is a poor measure of (\alpha)-NADH. By combination of preparative anion exchange chromatography and reversed-phase HPLC, (\alpha)-NADH can be prepared with (95-98 %) purity starting from (\beta)-NADH. Commercially available preparations contain only (80-90 % \alpha)-NADH. The anomerization kinetics of (\alpha) - and (\beta)-NADH has been investigated at pH 7.0 and 7.3 taking into account the irreversible conversion reactions of (\alpha) - and (\beta)-NADH. The (\mathrm{H}^{+})ion activity related anomerization rate constants (k_{1}(\alpha) to (\beta)) and (k_{2}(\beta) to (\alpha) ) have been found to be 170 and (29 \mathrm{~m}^{-1} \mathrm{~s}^{-1}), respectively, at pH 7.0 and 192 and (36 \mathrm{~m}^{-1} \mathrm{~s}^{-1}), respectively, at pH 7.3. The equilibrium constants (k_{1} / k_{2}) are 5.8 and 5.3 at pH 7.0 and pH 7.3, respectively. Absorption and fluorescence properties of (\alpha)-NADH preparations have been determined newly. At (25^{\circ} \mathrm{C}), maximum absorption was found at 260.1 and (346.6 \mathrm{~nm}), whereas at (37^{\circ} \mathrm{C}) the wavelengths were 260.1 and (345.3 \mathrm{~nm}). Maximum fluorescence excitation was found at (348 \mathrm{~nm}), and maximum emission was at (453 \mathrm{~nm}). c 1993 Academic Press, Inc.