Virulence in pathogenic bacteria is due in part to the action of two-component systems. However, in the human pathogen Mycobacterium tuberculosis, the molecular mechanisms underlying these systems are as yet unclear. In this study, MtrA was shown to contain a functional C-terminus and also to have Ca(2+) as its preferred cofactor for DNA binding. Further mutation experiments demonstrated that the C-terminus of MtrA was responsible for specific interactions with the target DNA motif and also with its partner protein, MtrB. The physical interaction between MtrA and MtrB inhibited DNA binding by MtrA. These findings yield critical information about the unique regulatory mechanisms of the essential MtrAB two-component system in this pathogen.