We have developed a separation system for N(epsilon)-(carboxyethyl)lysine (CEL) and N(epsilon)-(carboxymethyl)lysine (CML) by HPLC equipped with a styrene-divinylbenzene copolymer resin coupled with sulfonic group cation-exchange column and examined whether CEL is formed from proteins modified by glucose via the Maillard reaction. CEL was generated by incubating bovine serum albumin (BSA) with glucose, a reaction inhibited by aminoguanidine, but enhanced by phosphate. Although several aldehydes were detected during incubation of N(alpha)-acetyllysine with glucose, incubation of BSA with methylglyoxal alone generated CEL. These results indicate that methylglyoxal is responsible for CEL formation on protein in vitro.