Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45 amino-acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high-affinity complex formation involves multiple interactions between these colicins and TonB. As well as being essential for uptake and thus killing, the central domain also contributes to the formation of the immunity complex. Colicin D is normally secreted in association with the immunity protein and this complex involves two interactions: A major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the 4 helix of ImmD, which is on the opposite side of ImmD compared to the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity towards tRNA^Arg and a central domain which can form a complex with ImmD but which does not function in TonB-mediated transport.