Malate dehydrogenase (decarboxylating) (EC 1.1.1.39) was purified to near homogeneity from both a C3 plant, Solanum tuberosum, and a CAM plant, Crassula argentea. Sodium dodecyl sulfate-gel electrophoresis of both enzymes revealed an alpha,beta subunit composition with corresponding molecular mass assignments of 61,000 and 55,000 daltons. Isoelectric focusing under native conditions showed only one constituent malic enzyme form with an isoelectric point of 5.1. No evidence of additional isoenzymes was found. Urea isoelectric focusing showed the alpha subunit to be more acidic than the beta subunit. Peptide mapping by limited proteolysis with Staphylococcus aureus V-8 protease, trypsin, and endoproteinase Arg-C eliminated the possibility that a precursor-product relationship may have existed between the two subunits and demonstrated that they each possess unique primary sequences. Further support for this conclusion was obtained when significant differences in the contents of glutamic acid, isoleucine, and arginine were revealed by amino acid analysis of the isolated subunits. There was no apparent activity associated with the separated subunits (as resolved by urea-DEAE chromatography), but activity could be found in a reconstituted system, thereby indicating an (alpha,beta)n protomeric configuration. This is the first case where malic enzyme has been conclusively shown to be constructed from nonidentical subunits. This phenomenon has been observed only for the NAD malic enzyme isolated from plants.