The nature and composition of the unsaturated (monoenoic) oxo fatty acids in Cheddar cheese have been re-evaluated. Following isolation by column chromatography and silver ion HPLC, the fatty acids were identified in the form of their picolinyl ester derivatives by gas chromatography–mass spectrometry. For confirmation, they were analysed similarly following deuteration of the double bonds with deuterium and Wilkinson's catalyst. Fifteen fatty acids were identified fully, only five of which corresponded to those reported in an earlier study. Isomers with double bonds in positions 5, 7, 9 and 11–15 were identified, with oxo groups in positions 8, 9, 10, 13, 16 and 17, and with chain-lengths varying from C14 to C18, but with C18 predominating.