A 3.0-kb Hind III fragment of Thermus flavus AT-62 DNA cloned in Escherichia coli was shown to direct the synthesis of thermophilic malate dehydrogenase. The enzyme produced by the recombinant clone was purified by a relatively simple procedure of heat treatment and affinity chromatography nearly to homogeneity. The enzyme preparation had practically the same heat stability and kinetic and immunological properties as those of the enzyme prepared from T. flavus cells.