A thermostable 'phosphoenolpyruvate carboxylase' has been isolated from leaves of Zea mays different from phosphoenolpyruvate carboxylase (EC. 4.1.1.31) in that its optimum pH is 5.4, it does not liberate orthophosphate during the reaction, and it is inhibited by cyanide. The enzymic reaction has an optimum temperature of 70–75C and has been purified through steps including acidification to pH 4.6, heat treatment to 50C, and DEAE-cellulose and Sephadex G-200 column chromatography. Three fractions were active in the Sephadex eluate, but only fraction III was free from a thermostable acid phosphatase which catalyzes the liberation of orthophosphate from the substrate and the end product which is suggested to be a C4 phosphocarbonyl compound, although phosphohydroxypyruvate appears by either spontaneous or enzymic decarboxylation. The enzyme is assayed by the formation of a phenyl-hydrazone at 325 nm. The enzyme is localized and tightly bound in both the parenchyma bundle sheath and mesophyll chloroplasts, which are free from the thermostable acid phosphatase. Similar concentrations of the enzyme have been found in all plant species tested including C3 plants, ferns, bryophytes, algae, fungi, and even in calf liver. The enzyme must have considerable evolutionary significance.