The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33–239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33–239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33–239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter–TonB interaction that is mediated by the Ton box. Characterization of TonB(33–239) using double electron–electron resonance (DEER) demonstrates that a significant population of TonB(33–239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33–239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33–239) dimer is converted to a monomeric form, suggesting that a dimer–monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.