Mutant strains of yeast Saccharomyces cerevisiae lacking a functional F1-ATPase were found to grow very poorly under anaerobic conditions. A single amino acid replacement (K222 > E222) that locally disrupts the adenine nucleotide catalytic site in the beta-F1 subunit was sufficient to compromise anaerobic growth. This mutation also affected growth in aerated conditions when ethidium bromide (an intercalating agent impairing mtDNA propagation) or antimycin (an inhibitor of respiration) was included in the medium. F1-deficient cells forced to grow in oxygen-limited conditions were shown to lose their mtDNA completely and to accumulate Hsp60p mainly under its precursor form. Fluorescence microscopy analyses with a modified GFP containing a mitochondrial targeting presequence revealed that aerobically growing F1-deficient cells stopped importing the GFP when antimycin was added to the medium. Finally, after total inactivation of the catalytic alpha3beta3 subcomplex of F1, mitochondria could no longer be energized by externally added ATP because of either a block in assembly or local disruption of the adenine nucleotide processing site. Altogether these data strengthen the notion that in the absence of respiration, and whether the proton translocating domain (F0) of complex V is present or not, F1-catalysed hydrolysis of ATP is essential for the occurrence of vital cellular processes depending on the maintenance of an electrochemical potential across the mitochondrial inner membrane.