The multiprotein TonB system of Escherichia coli involves proteins in both the cytoplasmic membrane and the outer membrane. By a still unclear mechanism, the proton-motive force of the cytoplasmic membrane is used to catalyze active transport through high-affinity transporters in the outer membrane. TonB, ExbB, and ExbD are required to transduce the cytoplasmic membrane energy to these transporters. For E. coli, transport ligands consist of iron-siderophore complexes, vitamin B12, group B colicins, and bacteriophages T1 and ø80. Our experimental philosophy is that data gathered in vivo, where all known and unknown components are present at balanced chromosomal levels in the whole cell, can be interpreted with less ambiguity than when a subset of components is overexpressed or analysed in vitro. This chapter describes in vivo assays for the TonB system and their application.