Both α-crystallin from bovine eye lens and Hsp16.3 from Mycobacterium tuberculosis are members of the small heat shock protein family, They were preincubated at 100 C for 15 min and then cooled on ice immediately. The chaperone-like activities of preheated proteins were measured at 37 C using DTT-treated insulin B chains as substrates. Both preheated proteins exhibited greatly enhanced chaperone-like activities, accompanied with almost unchanged secondary structures and surface hydrophobicity but with a minor change in tertiary structures. The dramatically enhanced chaperone-like activities of preheated α-crystallln and Hsp16.3 may have resulted from the irreversible change in the tertiary structure as detected by near-UV CD spectra.