Comparative genomic studies based on whole genome DNA microarray have led to the identification of 16 regions of deletion (RDs) in Mycobacterium bovis BCG, which is currently used as a vaccine, with respect to Mycobacterium tuberculosis and five RDs with respect to M. bovis. RD1 is absent from all strains of BCG and Mycobacterium microti, whereas it is present in all virulent strains of M. tuberculosis and M. bovis [1]. The RD1 region in M. tuberculosis is 9455 bp long, and encompasses nine ORFs (Rv3871-Rv3879c). The 6-kDa early secretory antigenic target (ESAT-6) and culture filtrate protein-10 (CFP-10), expressed from the region of deletion-1 (RD1) of Mycobacterium tuberculosis H37Rv, are known to play a key role in virulence. In this study, we explored the thermodynamic and biochemical changes associated with the formation of the 1 : 1 heterodimeric complex between ESAT-6 and CFP-10. Using isothermal titration calorimetry (ITC), we precisely determined the association constant and free energy change for formation of the complex to be 2 · 10 7 m )1 and )9.95 kcalAE mol )1 , respectively. Strikingly, the thermal unfolding of the ESAT-6-CFP-10 heterodimeric complex was completely reversible, with a T m of 53.4°C and DH of 69 kcalAEmol )1 . Mixing of ESAT-6 and CFP-10 at any temperature below the T m of the complex led to induction of helical conformation, suggesting molecular recognition between specific segments of unfolded ESAT-6 and CFP-10. Enhanced biochemical stability of the complex was indicated by protection of ESAT-6 and an N-terminal fragment of CFP-10 from proteolysis with trypsin. However, the flexible C-terminal of CFP-10 in the complex, which has been shown to be responsible for binding to macrophages and monocytes, was cleaved by trypsin. In the presence of phospholipid membranes, ESAT-6, but not CFP-10 and the complex, showed an increase in a-helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced thermodynamic and biochemical stability, and loss of binding to phospholipid membranes. These features of complex formation probably determine the physiological role of ESAT-6, CFP-10 and ⁄ or the complex in vivo. The ITC and thermal unfolding approach described in this study can readily be applied to characterization of the 11 other pairs of ESAT-6 family proteins and for screening ESAT-6 and CFP-10 mutants.Abbreviations ANS, 8-anilinonapthalene-1-sulfonate; CFP-10, 10-kDa culture filtrate protein; DPC, dodecylphosphocholine; DSC, differential scanning calorimetry; ESAT-6, 6-kDa early secretory antigenic target; ESAT-6-CFP-10 complex, 1 : 1 complex of ESAT-6 and CFP-10; HSQC, heteronuclear single quantum correlation; ITC, isothermal titration calorimetry; Myr 2 PtdCho, dimyristoyl-DL-a-phosphatidylcholine; Ni ⁄ NTA, nickel ⁄ nitrilotriacetic acid; RD1, region of deletion 1; trCFP-10, truncated 10-kDa culture filtrate protein.