With a view to their use in the kinetic resolution of racemic non-natural amino acids, five variants of the enzyme L-phenylalanine dehydrogenase, the wild-type enzyme from Bacillus sphaericus and four active-site mutants, have been tested with a range of amino acids. In each case, the rates of reaction with 0.2 mM L-amino acid and with the racemic mixture at 0.4 mM were compared, so that the starting concentration of the active substrate was kept constant. Although the D-amino acids are not substrates, they were inhibitory in all cases. The extent of inhibition, however, varied greatly from compound to Compound and among the Mutants. With the N145L mutant and DL 4-O-Me-Phe, the equimolar D-enantiomer gave 83.2% inhibition, and with the wild-type enzyme there was 86.7% inhibition with racemic norleucine. By contrast, with these Same Substrates the N145V Mutant showed less than 9% and 24% inhibition respectively. The N145A mutant was selected for use with DL-4-Cl-Phe. The pH was decreased from the enzyme's optimum of 10.4 to 9.5 to minimise breakdown of the coenzyme NAD(+), and the coenzyme was recycled by molecular oxygen with the assistance of a commercial diaphorase. Reaction oil a 200 mu mole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 mu g N145A enzyme and 100 mu g diaphorase, was monitored by chiral HPLC. The L-isomer was removed to all extent of > 99% after 40 h, with the D-isomer peak undiminished. The pure D-isomer was isolated from the reaction Mixture in 85% overall yield after ion-exchange chromatography.