F1-ATPase assembly has been studied in human rho degrees cells devoid of mitochondrial DNA (mtDNA). Since, in these cells, oxidative phosphorylation cannot provide ATP, their growth relies on glycolysis. Despite the absence of the mtDNA-coded F0 subunits 6 and 8, rho degrees cells possessed normal levels of F1-ATPase alpha and beta subunits. This F1-ATPase was functional and azide- or aurovertin-sensitive but oligomycin-insensitive. In addition, aurovertin decreased cell growth in rho degrees cells and also reduced their mitochondrial membrane potential, as measured by rhodamine 123 fluorescence. Therefore, a functional F1-ATPase was important to maintain the mitochondrial membrane potential and the growth of these rho degrees cells. Bongkrekic acid, a specific adenine nucleotide translocator (ANT) inhibitor, also reduced rho degrees cell growth and mitochondrial membrane potential. In conclusion, rho degrees cells need both a functional F1-ATPase and a functional ANT to maintain their mitochondrial membrane potential, which is necessary for their growth. ATP hydrolysis catalyzed by F1 must provide ADP3- at a sufficient rate to maintain a rapid exchange with the glycolytic ATP4- by ANT, this electrogenic exchange inducing a mitochondrial membrane potential efficient enough to sustain cell growth. However, since the effects of bongkrekic acid and of aurovertin were additive, other electrogenic pumps should cooperate with this pathway.