F1-ATPase is inactivated by entrapment of MgADP in catalytic sites and , usinga mutant a3b3c complex of thermophilic F1-ATPase (aW463F/bY341W) and monitoring nucleotide binding by ¯uorescence quenching of an introduced tryptophan, we found that P i interfered with the binding of MgATP to F1-ATPase, but binding ofMgADPwas interferedwith to a lesser extent.