The stator function in ATP synthase was studied by a combined mutagenesis and fluorescence approach. Specifically, binding of δ-subunit to δ-depleted F1 was studied. A plausible binding surface on δ-subunit was identified from conservation of amino acid sequence and the high resolution NMR structure. Specific mutations aimed at modulating binding were introduced onto this surface. Affinity of binding of wild-type and mutant δ-subunits to δ-depleted F1 was determined quantitatively using the fluorescence signals of natural δ-Trp-28, inserted δ-Trp-11, or inserted δ-Trp-79. The results demonstrate that helices 1 and 5 in the N-terminal domain of the δ-subunit provide the F1-binding surface of δ. Unexpectedly, mutations that impaired binding between F1 and δ were found to not necessarily impair ATP synthase activity. Further investigation revealed that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced affinity of binding of δ-subunit to F1. The new data show that the stator is "overengineered" to resist rotor torque during catalysis.