After heating N-alpha-hippuryl-L-arginine (Hip-Arg) with varying amounts of lactose for 1-4 h at 100 degreesC, a previously unknown arginine derivative could be detected by RP-HPLC and UV-detection. Following semi-preparative isolation, the compound was unequivocally identified as 2-(2-benzoylamino-acetylamino)-5-[5-(3-hydroxypropyl)-4-oxo-imidazolon-2-yl]-L-ornithine (Hippuryl-PIO, Hip-PIO) by electrospray-time of flight-mass spectroscopy as well as one- and two-dimensional H-1- and C-13-nuclear magnetic resonance. Hip-PIO was exclusively formed during incubation of Hip-Arg with disaccharides containing a 1,4-glycosidic linkage. As reference for amino acid analysis, free PIO was synthesized starting from N-(tert-butoxycarbonyl)-L-arginine (t-Boc-Arg) via t-Boc-PIO and final hydrolysis with acetic acid. Preliminary evidence for the formation of protein-bound PIO, which proved to be acid-labile, was obtained using amino acid analysis with ninhydrin detection for enzymatic hydrolysates of casein samples which had been heated in the presence of lactose at 100degreesC. The ornithinoimidazolinone PIO represents a new type of post-translational protein modification formed during food processing, which might be responsible for the major part of arginine derivatisation in disaccharide-containing foods like milk.