Based on the potential benefits of cis-9, trans-11 conjugated linoleic acid (CLA) for human health there is a need to develop effective strategies for enhancing milk fat CLA concentrations. In this experiment, the effect of forage type and level of concentrate in the diet on milk fatty acid composition was examined in cows given a mixture of fish oil and sunflower oil. Four late lactation Holstein-British Friesian cows were used in a 4 × 4 Latin-square experiment with a 2 × 2 factorial arrangement of treatments and 21-day experimental periods. Treatments consisted of grass (G) or maize (M) silage supplemented with low (L) or high (H) levels of concentrates (65 : 35 and 35 : 65; forage : concentrate ratio, on a dry matter (DM) basis, respectively) offered as a total mixed ration at a restricted level of intake (20 kg DM per day). Lipid supplements (30 g/kg DM) containing fish oil and sunflower oil (2 : 3 w/w) were offered during the last 14 days of each experimental period. Treatments had no effect on total DM intake, milk yield, milk constituent output or milk fat content, but milk protein concentrations were lower (P < 0.05) for G than M diets (mean 43.0 and 47.3 g/kg, respectively). Compared with grass silage, milk fat contained higher (P < 0.05) amounts of C12:0, C14:0, trans C18:1 and long chain ≥ C20 (n-3) polyunsaturated fatty acids (PUFA) and lower (P < 0.05) levels of C18:0 and trans C18:2 when maize silage was offered. Increases in the proportion of concentrate in the diet elevated (P < 0.05) C18:2 (n-6) and long chain ≥ C20 (n-3) PUFA content, but reduced (P < 0.05) the amount of C18:3 (n-3). Concentrations of trans-11 C18:1 in milk were independent of forage type, but tended (P < 0.10) to be lower for high concentrate diets (mean 7.2 and 4.0 g/100 g fatty acids, for L and H respectively). Concentrations of trans-10 C18:1 were higher (P < 0.05) in milk from maize compared with grass silage (mean 10.3 and 4.1 g/100 g fatty acids, respectively) and increased in response to high levels of concentrates in the diet (mean 4.1 and 10.3 g/100 g fatty acids, for L and H, respectively). Forage type had no effect (P > 0.05) on total milk conjugated linoleic acid (CLA) (2.7 and 2.8 g/100 g fatty acids, for M and G, respectively) or cis-9, trans-11 CLA content (2.2 and 2.4 g/100 g fatty acids). Feeding high concentrate diets tended (P < 0.10) to decrease total CLA (3.3 and 2.2 g/100 g fatty acids, for L and H, respectively) and cis-9, trans-11 CLA (2.9 and 1.7 g/100 g fatty acids) concentrations and increase milk trans-9, cis-11 CLA and trans-10, cis-12 CLA content. In conclusion, the basal diet is an important determinant of milk fatty acid composition when a supplement of fish oil and sunflower oil is given.