FtsH is a membrane-bound ATP-dependent zinc-metalloprotease which proteolytically regulates the levels of specific membrane and cytoplasmic proteins that participate in diverse cellular functions, and which therefore might be of critical importance to a human pathogen such as Mycobacterium tuberculosis. As the substrates of MtFtsH in mycobacteria are not known, we examined whether recombinant MtFtsH could complement the lethality of a DeltaftsH3::kan mutation in Escherichia coli and elicit proteolytic activity against the known substrates of E. coli FtsH, namely heat shock transcription factor sigma(32) protein, protein translocation subunit SecY and bacteriophage lambdaCII repressor protein. The MtFtsH protein could not only efficiently complement lethality of DeltaftsH3::kan mutation in E. coli, but could also degrade all three heterologous substrates with specificity when expressed in ftsH-null cells of E. coli. These observations probably reveal the degree of conservation in the mechanisms of substrate recognition and cellular processes involving FtsH protease of M. tuberculosis and E. coli.