The effect of citrate on the structure and function of porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37) has been characterized. The native dimeric form of this enzyme is specifically activated by citrate in the NAD+ leads to NADH direction and inhibited by citrate in the NADH leads to NAD+ direction. It is proposed that citrate is bound at a regulatory site that is distinct from the catalytic site of the enzyme. In binding to this regulatory site, citrate greatly reduces the binding of NADH as determined by fluorescence titration and "Hummel-Dreyer"-type experiments, but does not diminish the binding of NAD+. As would be expected for an effector altering the equilibrium between two conformational forms of an enzyme, citrate favorably perturbs the equilibrium for the reaction in the direction of NAD+ reduction. Using [14C]citrate, the stoichiometry of citrate binding to mitochondrial malate dehydrogenase has been determined to be two equivalent sites per dimer, with a dissociation constant of 12.5 microM. In detailed kinetic studies, it has also been observed that activation by citrate abolishes (masks) the enzymatic activation induced by high concentrations of the substrate, L-malate. In addition, Hummel-Dreyer-type experiments indicate that less than a stoichiometric amount of NADH is bound to the enzyme under conditions of malate activation. These data are consistent with a previously suggested second "substrate" binding site proposed to explain the enzymatic activation observed at high concentrations of the substrate, L-malate (Telegdi, M., Wolfe, D. V., and Wolfe, R. G. (1973) J. Biol. Chem. 248, 6484-6489). This allosteric site may exist only on the enzyme conformation capable of binding NAD+.