When Ehrlich ascites carcinoma (EAC) cells and thymocytes were treated with uncoupler or rotenone in glucose-free medium, rapid ATP depletion was observed in both types of the cells. Oligomycin slowed down ATP loss in thymocytes, but not in EAC cells. Thus, mitochondrial ATP hydrolysis appears to be significant in deenergized thymocytes in contrast to EAC cells, in which other ATP consuming reactions were prevailing. Complete deenergization of mitochondria by uncoupler or rotenone in these cells resulted in inactivation of mitochondrial ATPase by 65-75%. The effect was observed after complete and rapid (20-30 s) disruption of the cells with detergent, Lubrol WX. ATPase was blocked by the specific inhibitor protein (IF1) as it was shown by the studies on reactivation of this enzyme. When respiration is blocked but ATP content is supported by glycolysis, mitochondrial ATPase is not suppressed by IF1, and maintains the energization of mitochondria. It is concluded that under complete de-energization of mitochondria IF1 significantly inhibits mitochondrial ATP hydrolysis and may slow down ATP loss in thymocytes and EAC cells.