α-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with α-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of α-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to α-crystallin during chaperone-like action. The first method involved the incubation of α-crystallin with ADH peptides at 48 o C for 1 h followed by separation and analysis of bound peptides. In the second method, α-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1 ,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 o C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH 4 0 - 6 0 ) as the α-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between α-crystallin and a synthetic peptide representing the binding site of ADH.