AbstractIt may be desirable to increase the level of conjugated linoleic acid (CLA) in milk as a health benefit in human nutrition. The purpose of this work was to separate the effects of linoleic and linolenic acids on CLA production in dairy cows and to determine to what extent endogenous synthesis contributes to cis-9, trans-11 CLA concentration in milk fat. Eight lactating cows and four non-lactating duodenal fistulated cows were used in a 4 ✕ 4 Latin-square design. All cows received a basal diet of grass silage that was supplemented with one of four concentrates, which were designed to differ in their linoleic and linolenic acid contents. The oil components of the concentrates were produced from mixtures of olive, linseed, rape, soya and sunflower oils to produce the four treatments: low linoleic/ low linolenic acid (LL), low linoleic/high linolenic acid (LH), high linoleic/low linolenic acid (HL) and high linoleic/ high linolenic acid (HH). Milk cis-9, trans-11 CLA contents were 0·8, 0·9, 0·9 and 1·1 g/100 g fatty acid methyl esters (P < 0·05) and yields were 5, 7, 7 and 8 g/day (P < 0·05) for the LL, LH, HL and HH treatments, respectively. The yields of trans-C18:1 fatty acids in milk were 19, 22, 21 and 23 g/day (P < 0·05), respectively. Taking the data for the cis-9, trans-11 CLA content and flow of duodenal fluid from the fistulated cows and representing this in terms of dietary intake by the lactating animals, the amounts of cis-9, trans-11 CLA produced in the rumen were calculated to be 0·8, 0·9, 1·2 and 1·1 g/day (P < 0·05) and for trans-C18:1 fatty acids 58, 58, 66 and 69 g/day (P < 0·05). Increasing linoleic and/or linolenic acids in the diet can increase the cis-9, trans-11 CLA content of cows’ milk. Only diets high in linoleic acid increased cis-9, trans-11 CLA production in the rumen. On all four diets, more than 80% of cis-9, trans-11 CLA in milk was produced endogenously by Δ9-desaturase from trans-11 C18:1 in the mammary gland. Cows on the same diet have different milk fat cis-9, trans-11 CLA concentrations that may be partially explained by differences in Δ9-desaturase activity between cows. Increasing the activity of Δ9-desaturase in the mammary gland may offer greater potential for enhancing the cis-9, trans-11 CLA content of milk fat than increasing cis-9, trans-11 CLA production in the rumen.