Since the water-insoluble crystallins of the lens may be the precursors of cataract, identifying the modifications that differentiate the water-insoluble from the water-soluble crystallins may provide the basis for understanding the chemistry leading to cataract. This investigation of the alpha-crystallins of the water-insoluble urea-soluble portion of 45-year-old normal clear lenses, isolated using gel filtration, ion exchange and reversed phase chromatography, has employed state-of-the-art mass spectrometric techniques to identify and locate the modifications of the water-insoluble alpha-crystallins. Modifications present in the isolated alpha-crystallins were identified by the molecular weights of the modified proteins, by the molecular weights of peptides produced by enzymatic digestion of the proteins, and by the fragmentation patterns produced by collisional activation of the peptides. Modifications that are either unique to the water-insoluble alpha-crystallins or are more prevalent in the water-soluble portion than in the water-soluble part include complete oxidation of the two Cys residues of alpha A-crystallin to form an intra-molecular disulfide bond, partial truncation at both the C-termini and N-termini of alpha A- and alpha B-crystallins, partial oxidation of Met residues to methionine sulfoxide, partial deamidation of several Asn and Gln residues, and evidence of peptide bond cleavage at some of the deamidated residues. Although many reactions have been proposed to contribute to the insolubility of crystallins, this compilation of in vivo post-translational modifications of water-insoluble alpha-crystallins delineates products that are actually present at levels of 5% or more. From these results, it is hypothesized that alpha-crystallin becomes water-insoluble following deamidation of various Asn and Gln residues which cause conformational changes leading to formation of an intra-molecular disulfide bond between the Cys residues of alpha A-crystallin.