Abstract A group of oligomycin-resistant mutants of Saccharomyces cerevisiae have been selected. Most are cytoplasmically inherited and have altered ATPase sensitivity in isolated mitochondria. Two mutants, resistant to growth on oligomycin, are not resistant when ATPase is tested in isolated mitochondria. One of these mutants shows normal Mendelian inheritance while the other is cytoplasmic. Studies on the isolated soluble ATPase enzyme from wild type cells show that the solubilized enzyme is not inhibited by oligomycin but the reconstituted enzyme complex is sensitive. ATPases from oligomycin-resistant mutants can also be separated into their component parts and subsequently reconstituted. The reconstituted enzyme complex resembles the mitochondrial-bound enzyme in its resistance to oligomycin inhibition. Preparation of hybrid reconstituted ATPase complexes from mixed components of the mutant and wild type enzymes shows that the resistance is contributed by the membrane fraction of the mutant ATPase. Thus, the mitochondrial DNA has been shown to direct the synthesis of a membrane component influencing the activity of the mitochondrial ATPase.