Crossline is one of the major advanced glycation end products resulting the reaction mixture of free amino group(s) such as epsilon-one in lysine with D-glucose in vitro. To study crossline formation on proteins in vitro and in vivo, polyclonal antiserum to the crossline hapten was prepared. This antiserum reacted with bovine and human serum albumin that had been modified by prolonged incubation with glucose as well as with crossline itself. Antisera did not react with unmodified serum albumin or the other Maillard-related compounds. Crossline was formed in a time-dependent manner when a mixture of six different proteins was incubated with glucose at pH 7.2 or 9.0. Crossline levels could be measured in rat lens proteins and the levels increased with age. The crossline content of lens proteins in diabetic rats was more than two-fold higher than that of age-matched controls. Results of this study suggest that most proteins containing advanced glycation end products have crossline-like structures. Measurement of crossline-like structures in biological specimens may provide an index of aging and of the development of diabetic complications.