Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive β3 sequence, ₇₃DRFSVNLDVKHFS₈₅ of human αB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the α crystallin core domain. Site-directed mutagenesis produced the mutant αB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous β3 sequences, ₃₂EKFEVGLDVQFFT₄₄ from Caenorhabditis elegans sHSP12.2 or ₆₉DKFVIFLDVKHFS₈₁ from αA crystallin, resulted in the mutant αB crystallins, CE1 and αA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using β_L crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with αA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human αB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive β3 sequence of the α crystallin core domain in αB crystallin for chaperone function.