The oxidative process in milk fat, resulting in spontaneous oxidized off-flavour (SOF), is commonly assumed to depend on contents of pro- and antioxidants in milk and availability of fatty acids acting as their substrate. An important antioxidant in milk is α-tocopherol whereas the most potent prooxidant is the metal ion copper. The separate effects of α-tocopherol, copper, and milk fatty acid profile, and their combined effect on SOF development were examined in milk from 44 multiparous cows fed different roughage types and different amounts of dietary, unsaturated fat. A clear association between concentrations of copper and poly-unsaturated fatty acids in milk and the risk for developing SOF was found. Heritability estimates suggest that occurrence of SOF is partly under genetic control which indicates that milk quality may be compromised if breeding bulls are selected that carry genotypes predisposing for milk prone to develop SOF.