Advanced glycation end products (AGEs) are protein modifications resulting from the chemical reaction between lysine and arginine residues in proteins, and carbonyl compounds, including glyoxal (GO) and methylglyoxal (MGO). Nε -carboxymethyllysine (CML) and Nε -carboxyethyllysine (CEL), formed by glycation from GO and MGO, are among the major AGEs in tissue proteins. Incubation with GO or MGO resulted in higher CML and CEL formation in the two cysteine residues containing aA-crystallin (aAC) than in the cysteine lacking aBcrystallin (aBC). Mass spectrometric data showed K70 and K166 to be heavily modified with CML and CEL in GO- and MGO-modified aAC. In silico analysis of the structure of aAC showed K70 and K166 to be proximal to C142. Mutation or reductive alkylation of cysteine residues in aAC significantly reduced CML and CEL formation. The addition of GSH or Nacetylcysteine enhanced CML and CEL formation in aBC. The introduction of a cysteine residue proximal to a lysine residue in aBC increased the CML and CEL accumulation. Our data showed that CML and CEL formation occurs through a hemithioacetal intermediate formed from the reaction between thiols and GO or MGO. Together, these results highlight a mechanism by which thiols influence protein AGE levels. In addition, CML and CEL are ligands for RAGE, a receptor for AGEs, which has been implicated in several aging and diabetesassociated diseases. Therefore, further understanding of the biosynthesis of CML and CEL could lead to the development of new therapies against those diseases.