αA-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the αA-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knock-in mice expressing the arginine 49 to cysteine mutation (R49C) in αA-crystallin (αA-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four-generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. αA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, αA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type litter-mates did not show these abnormalities. Lens fiber cells of αA-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a 5-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo bromodeoxyuridine labeling did not decline. The αA-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble αA-crystallin and αB-crystallin and a surprising increase in expression of cytoplasmic α-crystallin, whereas no changes in β-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between γA-crystallin and lens substrate proteins in the heterozygous knock-in lenses. To our knowledge this is the first knock-in mouse model for a crystallin mutation causing hereditary human cataract and establishes that αA-R49C promotes protein insolubility and cell death in vivo.