The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane, derived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB. This process requires direct physical contact between the outer membrane receptor and TonB. Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors as being essential for productive energy transduction. In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB. Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact. This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors. This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained Gin-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction. These studies indicated that the final 48 residues of TonB were essential to this physical interaction. This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction. Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex. The effects of amino acid substitutions at several other TonB sites were also examined.