•Formation pathways of 2-alkyl-3-hydroxypyridines in foods are described.•Main precursors resulted to be 4,5-epoxy-2-alkenals and 2,4-alkadienals.•Hydroxypyridines are produced by cyclization of precursors induced by ammonia.•Hydroxypyridines are formed when oxidized oils are heated with ammonia.•Hydroxypyridines are generated as a consequence of the lipid oxidation pathway. Pyridines are produced during food processing and are important flavor compounds. In spite of that, their formation pathways are still poorly understood, in particular those related to 3-hydroxypyridines. In an attempt to fill this gap, this study describes, for the first time, precursors and reaction pathways leading to 3-hydroxypyridine formation. 3-Hydroxypyridines are produced by reaction of lipid-derived reactive carbonyls and ammonia-producing compounds and were studied by using gas chromatography coupled to mass spectrometry. Their main precursors resulted to be 4,5-epoxy-2-alkenals and 2,4-alkadienals. 3-Hydroxypyridines were produced at temperatures higher than 100 °C, at slightly basic pH values, and with an activation energy of about 50 kJ/mol. A reaction pathway that explains their formation in the course of the lipid oxidation pathway is proposed. The role of lipid oxidation on the production of 3-hydroxypyridines was confirmed by studying their formation in oxidized linseed and menhaden oils heated in the presence of glutamine.