Abstract The supernatant and mitochondrial forms of malate dehydrogenase ( l -malate:NAD oxidoreductase, EC 1.1.1.37) have been purified from Drosophila virilis Texmelucan (1801.1). The mitochondrial enzyme was judged homogeneous by ultra-centrifugal and electrophoretic criteria and has an s 20,w of 4.0 S. The molecular weights of both enzymes was estimated to be 68 000 by gel filtration on calibrated columns. The mitochondrial and supernatant forms of malate dehydrogenases could readily be differentiated with respect to inhibition by oxaloacetate, thermolability and reactivity with coenzyme analogs. A rabbit antiserum directed against Drosophila virilis Texmelucan mitochondrial malate dehydrogenase reacted strongly with this enzyme as judged by inhibition of enzyme activity and by double diffusion in agar. No cross-reaction was observed with the supernatant malate dehydrogenase. A comparison of the supernatant and mitochondrial malate dehydrogenases of Drosophila virilis Texmelucan with the corresponding enzymes from Drosophila melanogaster Oregon K revealed differences in immunological properties, in inhibition by substrate and in thermolability.