Cataract is a common cause of childhood blindness worldwide. alpha-crystallin, which is comprised of two homologous subunits, alphaA- and alphaB-crystallin, plays a key role in the maintenance of lens transparency. Recently, we have identified a missense mutation in alphaB-crystallin that changes the proline residue at codon 20 to a serine residue (P20S) in a large Chinese family with autosomal dominant posterior polar congenital cataract. To explore the molecular mechanism by which the P20S mutation causes cataract, we examined the quaternary structure, subunit exchange and chaperone activity of the reconstituted heteroaggregates of alpha-crystallins containing wild type (WT) alphaA in combination with either WT-alphaB- or mutant alphaB-crystallin, respectively. Compared with heteroaggregates of WT-alphaA and WT-alphaB, heteroaggregates containing WT-alphaA and mutant alphaB showed nearly the same molecular mass, but the subunit-exchange rate and chaperone activity were decreased markedly. In human lens epithelial cells, unlike WT-alphaB-crystallin, the P20S mutant protein showed abnormal nuclear localization, and unusual ability to trigger apoptosis. These results suggest that the changes in the structure and function of the alpha-crystallin complex and cytotoxicity are vital factors in the pathogenesis of congenital cataract linked to the P20S mutation in the alphaB-crystallin.