Inactivation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by selective modification of an active center histidine residue with the reagent iodoacetamide has been further investigated to examine the existence of and the enzymatic activity of a hybrid (half)-modified dimer. The loss of enzymatic activity during iodo(1-14C) acetamide modification is linear with 14C incorporation. Enzyme was modified to various extents and the reaction was quenched. Microzonal electrophoresis was performed to separate native dimeric enzyme, hybrid-modified enzyme, and doubly modified enzyme. The distribution of each species was quantitated by scanning densitometry. The distribution generated throughout the time course of inactivation indicates that both subunits are modified independently and at the same rate. It is apparent that the hybrid-modified dimer contributes one-half of the enzymatic activity of a native dimer in the standard assay. Kinetic studies were performed and the results indicate that there is no apparent change in kinetic parameters between a subunit of the native dimer and the active subunit in the hybrid-modified dimer. Dissociation and reassociation of a mixture of native enzyme and doubly-iodoacetamide-modified enzyme indicates that there is no preferential association of a modified subunit with another modified subunit, or of a native subunit with another native subunit, but rather, association is random with respect to native and iodoacetamide-modified subunits.