In total, 20 multiparous Holstein-Friesian dairy cows received one of four diets in each of four periods of 28-day duration in a Latin square design to test the hypothesis that the inclusion of lucerne in the ration of high-yielding dairy cows would improve animal performance and milk fatty acid (FA) composition. All dietary treatments contained 0.55 : 0.45 forage to concentrates (dry matter (DM) basis), and within the forage component the proportion of lucerne (Medicago sativa), grass (Lolium perenne) and maize silage (Zea mays) was varied (DM basis): control (C)=0.4 : 0.6 grass : maize silage; L20=0.2 : 0.2 : 0.6 lucerne : grass : maize silage; L40=0.4 : 0.6 lucerne : maize silage; and L60=0.6 : 0.4 lucerne : maize silage. Diets were formulated to contain a similar CP and metabolisable protein content, with the reduction of soya bean meal and feed grade urea with increasing content of lucerne. Intake averaged 24.3 kg DM/day and was lowest in cows when fed L60 (P<0.01), but there was no effect of treatment on milk yield, milk fat or protein content, or live weight change, which averaged 40.9 kg/day, 41.0, 30.9 g/kg and 0.16 kg/day, respectively. Milk fat content of 18:2 c9 c12 and 18:3 c9 c12 c15 was increased (P<0.05) with increasing proportion of lucerne in the ration. Milk fat content of total polyunsaturated fatty acids was increased by 0.26 g/100 g in L60 compared with C. Plasma urea and β-hydroxybutyrate concentrations averaged 3.54 and 0.52 mmol/l, respectively, and were highest (P<0.001) in cows when fed L60 and lowest in C, but plasma glucose and total protein was not affected (P>0.05) by dietary treatment. Digestibility of DM, organic matter, CP and fibre decreased (P<0.01) with increasing content of lucerne in the diet, although fibre digestibility was similar in L40 and L60. It is concluded that first cut grass silage can be replaced with first cut lucerne silage without any detrimental effect on performance and an improvement in the milk FA profile, although intake and digestibility was lowest and plasma urea concentrations highest in cows when fed the highest level of inclusion of lucerne.