The FtsH (HflB) protein of Escherichia coli is integrated into the membrane with two N-terminally located transmembrane segments, while its large cytoplasmic domain is homologous to the AAA family of ATPases. The previous studies on dominant negative ftsH mutants raised a possibility that FtsH functions in multimeric states. We found that FtsH was eluted at fractions corresponding to a larger molecular weight than expected from monomeric structure in size-exclusion chromatography. Moreover, treatment of membranes or their detergent extracts with a cross-linker, dithiobis(succinimidyl propionate), yielded cross-linked products of FtsH. To dissect possible FtsH complex, we constructed an FtsH derivative with c-Myc epitope at its C terminus (FtsH-His6-Myc). When membranes prepared from cells in which FtsH-His6-Myc was overproduced together with the normal FtsH were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His6-Myc without the tag were cross-linked with the tagged FtsH protein. Co-immunoprecipitation experiments confirmed the interaction between the FtsH molecules. To identify regions of FtsH required or sufficient for this interaction, we constructed chimeric proteins between FtsH and EnvZ, a protein with a similar topological arrangement, by exchanging their corresponding domains. We found that only the FtsH-EnvZ hybrid protein with an FtsH-derived membrane anchoring domain and an EnvZ-derived cytoplasmic domain caused a dominant ftsH phenotype and was cross-linked with FtsH. We suggest that the N-terminal transmembrane region of FtsH mediates directly the interaction between the FtsH subunits.