Bacterial condensin MukBEF is essential for global folding of the Escherichia coli chromosome. MukB, a SMC (structural maintenance of chromosome) protein, comprises the core of this complex and is responsible for its ATP-modulated DNA binding and reshaping activities. MukF serves as a kleisin that modulates MukB–DNA interactions and links MukBs into macromolecular assemblies. Little is known about the function of MukE. Using random mutagenesis, we generated six loss-of-function point mutations in MukE. The surface mutations clustered in two places. One of them was at or close to the interface with MukF while the other was away from the known interactions of the protein. All loss-of-function mutations affected focal localization of MukBEF in live cells. In vitro, however, only some of them interfered with the assembly of MukBEF into a complex or the ability of MukEF to disrupt MukB–DNA interactions. Moreover, some MukE mutants were able to join intracellular foci formed by endogenous MukBEF and most of the mutants were efficiently incorporated into MukBEF even in the presence of endogenous MukE. These data reveal that focal localization of MukBEF involves other activities besides DNA binding and that MukE plays a central role in them.