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Cells of the yeast Saccharomyces cerevisiae could be depleted of their intramitochondrial ATP by culturing on glucose in the presence of antimycin A, which prevents production of ATP in mitochondria, along with bongkrekic acid, which prevents transport of ATP from the cytosol into mitochondria. Alternatively, the depletion could be achieved by culturing respiration-deficient mutants in the presence of bongkrekic acid. The depleted cells of the respiration-deficient mutant did not grow on glucose in a synthetic medium and growth for a few generations was made possible by adding peptone, yeast extract or some amino acids into the medium. The depleted cells did not differ from control cells in their content of amino acids, proteins, nucleic acids and major phospholipids and had preserved the ability to carry on protein and nucleic acid syntheses and to mate to other cells. No conspicuous cytological differences were found between the control and depleted cells. After culturing in a semi-synthetic medium in the presence of bongkrekic acid the cells of the respiration-deficient mutant exhibited almost no cytochrome c in their spectra and their azide-sensitive ATPase activity was drastically reduced. The results suggest that intramitochondrial syntheses of some low-molecular compounds as well as import and/or assembly of some cytoplasmically synthesized mitochondrial proteins into mitochondria may be impaired in cells lacking intramitochondrial ATP and this may be responsible for their inability to grow and multiply.