The DevR-DevS two-component system of Mycobacterium tuberculosis mediates bacterial adaptation to hypoxia, a condition believed to be associated with the initiation and maintenance of dormant bacilli during latent tuberculosis. The activity of the Rv3134c-devRS operon was studied in M. tuberculosis using several transcriptional fusions comprised of promoter regions and the gfp reporter gene under inducing and aerobic conditions. Aerobic transcription was DevR independent, while hypoxic induction was completely DevR dependent. The hypoxia transcriptional start point, T(H), was mapped at -40 bp upstream of Rv3134c. In contrast, the divergently transcribed Rv3135 gene was not induced under hypoxic conditions. DNase I footprinting and mutational analyses demonstrated that induction required the interaction of DevR-P with binding sites centered at bp -42.5 and -63.5 relative to T(H). Binding to the distal site (D) was necessary to recruit another molecule of DevR-P to the proximal site (P), and interaction with both sequences was essential for promoter activation. These sites did not bind to either unphosphorylated or phosphorylation-defective DevR protein, which was consistent with an essential role for DevR-P in activation. Phosphorylated DevR also bound to three copies of the motif at the hspX promoter. The Rv3134c and hspX promoters have a similar architecture, wherein the proximal DevR-P binding site overlaps with the promoter -35 element. A model for the likely mode of action of DevR at these promoters is discussed.