Cytoplasmic and mitochondrial malate dehydrogenases from pig and chicken were studied by chemical modification of amino groups, hybridization of immobilization. Determination of thermal stability was used to characterize the different species. Modification of amino groups was found to decrease thermal stability especially when neutralization of the positive charges occurred. Decreased thermal stability correlated with decreased reassociation of immobilized monomers and modification in the monomeric state completely inhibited reassociation. Thus some lysines seem to be implicated within the subunit contacts. Active monomers of the mitochondrial forms as demonstrated earlier (Jürgensen, S.R., Wood, D.C., Mahler, J.C. and Harrison, J.H. (1981) J. Biol Chem. 256, 2383-2388) were found to display unaltered kinetic properties. From hybridizations the mechanism of thermal denaturation of malate dehydrogenases was concluded to contain a rate-limiting cooperative transaction of both monomers within the dimer, as was found earlier for tetrameric lactate dehydrogenase (Müller, J. (1981) Biochim. Biophys. Acta 669, 210-215 and Müller, J. and Klein, C. (1981) Biochim. Biophys. Acta 671, 38-41).