A simple and sensitive spectrophotometric method for the determination of D-glutamate has been developed. The method is based on the racemization of D-glutamate with glutamate racemase (EC 5.1.1.3) and the simultaneous dehydrogenation of L-glutamate with L-glutamate dehydrogenase (EC 1.4.1.3). NADH produced was determined either by the measurement of absorbance at 340 nm or by the colorimetric measurement of a formazan formed at 515 nm. A linear relationship has been obtained between the absorbance and the amount of D-glutamate (0.01-0.2 μmol). Enantioselective determination was also possible by measuring L-glutamate with L-glutamate dehydrogenase before the addition of glutamate racemase.