Mild trypsin digestion of isolated bovine-heart mitochondrial F 1 -ATPase removed the first 15 residues from the N-terminus of subunit α under conditions in which other F 1 subunits were apparently untouched. When the trypsinized F 1 (TF 1 ) was reconstituted with the F 0 sector in the mitochondrial membrane (USMP), the ATP hydrolase activity acquired oligomycin sensitivity but ATP hydrolysis was decoupled from proton pumping. TF 1 added to USMP did not block the proton channel in F 0 as the native F 1 did. AMP-PNP inhibited proton conductivity in reconstituted F 1 -USMP but this effect was lost in reconstituted TF 1 -USMP. These results indicate that the N-terminus of the F 1 α subunit plays a critical role in the conformational communication between F 1 and F 0 .