The substrate specificity of maleate hydratase from Arthrobacter sp. strain MCI2612 was examined with maleate and its derivatives. Maleate hydratase was shown to catalyze the hydration of maleate, chloromaleate, bromomaleate, and citraconate. Water was added trans to chloromaleate and bromomaleate to synthesize the (-)-erythro-beta-substituted derivatives of D-malate. (R)-(-)-Citramalate was synthesized from citraconate by using maleate hydratase. Many organic acids such as acetylenedicarboxylate, L(+)-, D(-)-, and meso-tartarate, and cis-, trans-epoxysuccinate inhibited competitively the formation of D-malate from maleate.