Exposure of purified mitochondrial F0F1 ATP synthase to H2O2 resulted in a marked inhibition of the ATPase activity, irrespective of the purification procedure used and of the incorporation of the enzyme complex into phospholipid vesicles. The inactivation appeared consequent to oxidative modifications of the F1 moiety, whereas damage to the F0 sector, leading to low enzyme activity through impaired binding with F1, seemed not to occur. In fact, when H2O2-inactivated complex was deprived of F1, no loss of the capacity of the F0 sector thus obtained to properly reassemble with untreated purified F1 was apparent, because the resulting enzyme complex showed full activity and oligomycin sensitivity. On the contrary, the exposure of the isolated components F1 or F0 to H2O2, followed by reassembly with untreated F0 and F1 respectively, resulted in both cases in lower catalytic activity of the reconstituted complexes, whereas low oligomycin sensitivity was exhibited only in the case of F0 treatment, suggesting the inactivation in this case as due to oxidative modifications leading to impaired binding with F1.