L-Hydrazinosuccinate has been reported to be a slow- and tight-binding inhibitor of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and to interact with the enzyme via a reaction of two consecutive steps. The present work examined the effects of D-hydrazinosuccinate on the same enzyme for comparison. D-Hydrazinosuccinate showed a potent inhibition in a slow-binding manner: transamination became slower with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and D-hydrazinosuccinate, while the reaction was initially very slow and became faster with time when the enzyme was preincubated with the inhibitor before the initiation of reaction. Analysis of the time-course of interaction of the enzyme with D-hydrazinosuccinate suggested a reversible single-step reaction mechanism and gave an inhibition constant of approx. 3 nM, in contrast to the two-step mechanism, and a much lower inhibition constant of 0.2 nM for L-hydrazinosuccinate. Comparison of the rate constants for the reaction steps in the interaction of the enzyme with D- and L-enantiomers confirmed that the difference in the reaction mechanism was mainly responsible for the stronger inhibition by the L-enantiomer. Spectral studies showed that D- and L-hydrazinosuccinate both produced complexes with the enzyme probably in the form of aldimine, and thereafter only the complex with L-hydrazinosuccinate further changed to another species more slowly, consistent with the two-step mechanism. The configuration of the hydrazino group is therefore crucial for the conversion of aldimine complexes to more tightly bound complexes.