ATP hydrolysis by the isolated F(1)-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3). The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F(1)-ATPase). The ATP hydrolysis activity of a load-free ensemble of F(1) with 12 residues deleted decreased to 24% of the control. EF(1) with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF(1) when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF(1) (full-length gamma) to 34 kJ/mol for EF(1)(gamma-12). These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.