Beta-citryl-L-glutamate (beta-CG) concentration was determined by HPLC during the differentiation of bovine lens epithelial cells into lens fiber cells in culture. beta-CG increased from 1 to 4 weeks of culture and then decreased slightly, while alpha-crystallin, a marker of lens cell differentiation, increased rapidly 4 weeks after the culture and continued to increase gradually until week 11. In addition, the localization of beta-CG was immunohistochemically examined using anti-beta-CG antibody. Cells around lentoid bodies were stained with anti-beta-CG antibody, whereas cells in the bodies were stained strongly with anti-gamma-crystallin antibody. These findings suggest that beta-CG accumulated immediately before the differentiation of the bovine lens epithelial cells into lens fiber cells and may play a role in regulating the differentiation of lens cells.