To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of δ-subunit to Escherichia coli F1-ATPase. The signal used is that of the natural tryptophan at residue δ28, which is enhanced by 50% upon binding of δ-subunit to α3β3γε complex. Kd for δ binding is 1.4 nM, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for δ binding was detected. The δW28L mutation increased Kd to 4.6 nM, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of δ-depleted F1. The αG29D mutation reduced Kd to 26 nM, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of δ to F1. Results with the two mutants illuminate how finely balanced is the stator resistance function. δ' fragment, consisting of residues δ1-134, bound with the same Kd as intact δ, showing that, at least in absence of Fo subunits, the C-terminal domain of δ contributes zero binding energy. Mg2+ ions had a strong effect on increasing δ binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced δ binding affinity, suggesting the involvement of protonatable side-chains in the binding site.