Three forms of NAD-dependent alcohol dehydrogenase have been characterized in yeast by their heat sensitivity, their specificity and their electrophoretic patterns. Thermal stability increases in the following order: alcohol dehydrogenase I (fermentative enzyme), alcohol dehydrogenase II (oxidative enzyme), alcohol dehydrogenase III (mitochondrial enzyme). Work with isolated mitochondria shows that alcohol dehydrogenase III is the only form of alcohol dehydrogenase present in these organelles. Starch gel electrophoresis of alcohol dehydrogenase III reveals an active zone of slow migration which consists of five sub-bands. The relative activity of these five sub-bands varies with the conditions of growth. Mitochondrial alcohol dehydrogenase represents never more than 10% of the total cellular alcohol dehydrogenase. Information for its biosynthesis seems to be located in nucleic DNA. The mitochondrial enzyme shows a high affinity for alcohols with a double bond conjugated to the alcohol function.