Abstract The action of crystalline fumarate hydratase (EC 4.2.1.2) on the three isomers of tartrate was investigated. Only the unnatural (—)-tartrate was found to be attacked by the enzyme. In the reaction mixture, oxalacetate and pyruvate were detected chromatographically. Their 2,4-dinitrophenylhydrazones could be detected by paper chromatography. The amino acids obtained by the reduction of the hydrazones were identified chromatographically with aspartic acid and alanine. The presence of oxalacetate and pyruvate in the reaction mixture could be confirmed by the action of malate dehydrogenase and lactate dehydrogenase. However, the formation of pyruvate was very slow at the beginning of the reaction. Thus, it was assumed that oxalacetate is the primary product and that pyruvate is formed by nonenzymatic decarboxylation of oxalacetate. A stoichiometric relationship was established between the disappearance of (—)-tartrate and the formation of keto acids. From the above results, the reaction catalyzed was assumed to be the dehydration of (—)-tartrate. This reaction satisfies the requirements of stereospecificity of the dehydration of l-malate by fumarate hydratase.