A critical point of interaction between F1 and F0 in the bacterial F1F0-ATP synthase is formed by the α and δ subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the δ subunit forms a 6 α-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between δ and F1 is provided by the interaction between the N-terminal 22 residues of the α- and N-terminal domain of the δ subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the δ-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the α subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of α-subunit residues with and without δ N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the δ subunit is formed by α helices 1 and V. NOE cross-peak patterns in 2D l2C/12C-filtered NOESY spectra of the l3C-labeled δ-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the α-subunit N-terminal peptide are folded as an α helix when bound to δ N-terminal domain. On the basis of intermolecular contacts observed in 12C/ l3C-filtered NOESY experiments, we describe structural details of the interaction of the δ-subunit N-terminal domain with the α-subunit N-terminal α helix.