Maltosine (6-(3-Hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine) is formed in the late stage of the Maillard reaction. Quantitative data on the occurrence in food of this metal-chelating 3-hydroxy-4(1H)pyridinone are not available. In the present study, the formation of the compound was studied first in model systems simulating bread crust and crumb. Maltosine was formed predominantly in the presence of di- and oligosaccharides and glycosylated isomaltol derivatives under conditions resembling crust formation. Disaccharide derivatives with β-glycosidic bonds were less potent precursors than derivatives with α-glycosidic bonds. Moreover, a method was developed for the quantification of maltosine in food. The compound was released from proteins by enzymatic hydrolysis due to artefact formation during acid hydrolysis. [13C6,15N2] labelled maltosine was synthesized and utilized as an internal standard, and the analysis of 62 commercial food samples was performed by HPLC–MS/MS in the multiple reaction monitoring mode. Maltosine was present predominantly in bread samples (0.1–4.2 mg/kg). The highest concentrations of the compound were found in the crust of wheat bread (up to 19.3 mg/kg), representing a maximal lysine modification of 0.4 %. The concentrations of maltosine in most food samples reached approximately 10 % of the concentrations of the advanced glycation end product pyrraline. From these data, an intake of maltosine of 1–2 mg per day can be expected from the diet.