Background. αA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the αA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family. Methods. This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C αA-crystallin. Mice carrying the neo r gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for αA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared. Results. By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 μm from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49C neo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neo r gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C αA-crystallin protein. Conclusion. It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the αA-crystallin mutation and rapidly leads to lens cell pathology in vivo.