α-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, α-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major αA- and αB-subunits, α-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, αAins-crystallin. This polypeptide is identical to normal αA-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat αA- and αAins-crystallin in Escherichia coli. The multimeric particles formed by αAins are larger and more disperse than those of αA, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with αA, the αAins-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of βlow-crystallin. Our experiments indicate that αAins-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant αAins-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.