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 α3β3 hexagon of F1. Proteolysis and cross-linking had suggested that N-terminal residues of a subunit are involved in binding δ. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of α ("αN1-22") binds specifically to isolated wild-type δ subunit with high affinity (Kd = 130 nM), accounting for a major portion of the binding energy when δ-depleted F1 and isolated δ bind together (Kd = 1.4 nM). Stoichiometry of binding of αN1-22 to δ at saturation was 1/1, showing that in intact F1F0-ATP synthase only one of the three α subunits is involved in δ binding. When αN1-22 was incubated with δ subunits containing mutations in helices 1 or 5 on the F1-binding face of δ, peptide binding was impaired as was binding of δ-depleted F1. Residues α6-18 are predicted to be helical, and a potential helix capping box occurs at residues α3-8. Circular dichroism measurements showed that αN1-22 had significant helical content. Hypothetically a helical region of residues αN1-22 packs with helices 1 and 5 on the F1-binding face of δ, forming the α/δ interface.