A Ca2+-dependent transglutaminase (EC 2.3.2.13) has been demonstrated in the eye lenses of several mammalian species [Lorand, L., Hsu, L. K. M., Siefring, G. E., Jr., & Rafferty, N. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1356-1360]. Using [3H]methylamine as a convenient probe for transglutaminase activity, we have explored the action of this enzyme in the bovine eye lens. We could characterize the glutamine residues acting as acyl-donor sites in three beta-crystallin chains, which are the only substrates for lens transglutaminase among the various lens-specific structural proteins, the crystallins. A single glutamine was found to bind [3H]methylamine in each of these three chains: glutamine -9 in beta Bp (beta B2), glutamine -21 in beta B3, and glutamine -23 or -24 in beta A3. The four glutamines are all located in the NH2-terminal regions, which presumably extend from the compact two-domain structure of the beta-crystallin chains. It was, moreover, established that several components of the lens cytoskeleton are substrates for transglutaminase.