The stator in F1F0-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b2δ subunit complex comprises the stator, bound to subunit a in F0 and to the α3β3 hexagon of F1. To quantitatively characterize binding of b subunit to the F1 α3β3 hexagon, we developed fluorimetric assays in which wild-type F1 or F1 enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b ST34-156). With five different F1 enzymes, K d(bST34-156) ranged from 91 to 157 nM. Binding was strongly Mg2+-dependent; in EDTA buffer, Kd(b ST34-156) was increased to 1.25 μM. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of δ subunit to δ-depleted F1. The apparent K d(bST34-156) for this effect was increased from 150 nM in Mg2+ buffer to 1.36 μM in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F1 contributes to stator resistance and emphasizes the importance of Mg2+ in stator interactions.