The stable isotope ratios ( ¹³ C/ ¹² C, ¹⁵ N/ ¹⁴ N, ¹⁸ O/ ¹⁶ O, D/H) of animal feed and milk were investigated, considering cows stabled in two farms and fed with diets made up of different kinds of C ₃ plants and different amounts of maize. Maize was characterised by δ ¹³ C, δ ¹⁸ O and δ D values significantly higher than those of the C ₃ plants, while, for the C ₃ plants, Festuca arudinacea had significantly higher content of ¹³ C and ¹⁵ N. The δ ¹³ C and δ ¹⁸ O values of the overall diet and the δ ¹³ C of milk casein and lipids were shown to be significantly correlated with the percentage of maize in the animal diet. On the other hand, the δ ¹⁸ O values of milk water and the δ ¹⁸ O, δ D and δ ¹⁵ N values of casein were shown to be only slightly influenced by the amount of maize in the feed, being probably more closely correlated with the geo‐climatic and pedological characteristics of the area of origin and with the presence of fresh plant or silage in the ration. The δ ¹³ C value of casein was shown to be a suitable parameter for evaluating the amount of maize in the diet: each 10% increase in the maize content corresponded to a shift of 0.7‰ to 1.0‰ in the δ ¹³ C of casein. A threshold value of −23.5‰ for δ ¹³ C in milk casein, above which it is not possible to exclude the presence of maize in the diet, was suggested. The results obtained could be useful for determining mislabelling of dairy products declared to have been produced by pastured animals or of PDO cheeses with an established amount of maize in the diet and for verifying the unpermitted addition of exogenous components to milk. Copyright © 2008 John Wiley & Sons, Ltd.