Small heat shock proteins (sHSPs), as one important subclass of molecular chaperones, are able to specifically bind to denatured substrate proteins rather than to native proteins, of which their substrate-binding sites are far from clear. Our previous study showed an overlapping nature of the sites for both hydrophobic probe 1,1'-Bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding and substrate binding in Mycobacterium tuberculosis Hsp16.3 [X. Fu, H. Zhang, X. Zhang, Y. Cao, W. Jiao, C. Liu, Y. Song, A. Abulimiti, Z. Chang, A dual role for the N-terminal region of M. tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins, J. Biol. Chem. 280 (2005) 6337-6348]. In this work, two bis-ANS binding sites in Hsp16.3 were identified by a combined use of reverse phase HPLC, mass spectroscopy and N-terminal protein sequencing. One site is in the N-terminal region and the other one in the N-terminus of alpha-crystallin domain, both of which are similar to those identified so far in sHSPs. However, accumulating data suggest that these two sites differentially function in binding substrate proteins. With regard to this difference, we proposed a two-step mechanism by which Hsp16.3 binds substrate proteins, i.e., substrate proteins are recognized and initially captured by the N-terminal region that is exposed in the dissociated Hsp16.3 oligomers, and then the captured substrate proteins are further stabilized in the complex by the subsequent binding of the N-terminus of alpha-crystallin domain.