Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in alphaB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alphaA-crystallin. To understand the significance of the RS-2 region in interactions between alphaA- and alphaB-crystallins, W60R, F61N, and S66G mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. W60R and S66G mutations increased the oligomeric size of alphaB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alphaBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alphaBF61N was marginally lower than that of alphaB, whereas the fluorescence intensity of alphaBW60R decreased by 40% compared with that of alphaB. The relative availability of hydrophobic sites in the mutants was in the following order: alphaBS66G >> alphaB = alphaBF61N = alphaBW60R. The far-UV CD profiles for the wild type and alphaB-crystallin mutants indicated no significant changes in their secondary structures, except for alphaBS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alphaBW60R and alphaBF61N were nearly similar to that of wild type alphaB. On the other hand, alphaBS66G beyond 270 nm exhibited a signature completely different from that of wild type alphaB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alphaB- and alphaA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alphaB- and alphaA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alphaB-crystallin and subunit interaction between alphaB- and alphaA-crystallins.