A partially balanced changeover design experiment was undertaken to evaluate the effects of level of fish oil inclusion in the diet on milk fat concentration and composition when offered to 50 lactating dairy cows in early lactation, which were offered either 5 or 10 kg concentrates/cow/day. Concentrates were formulated to contain similar concentrations of crude protein (CP), effective rumen degradable protein (ERDP) digestible undegradable protein and starch and to provide 0 (T0), 150 (T150), 300 (T300) or 450 (T450) g fish oil/cow/day or 300 g (T300B) fish oil/cow/day from a commercial fish oil premix. All animals were offered 5 kg treatment concentrate in two equal feeds through the in-parlour feeder at each milking. Additionally, cows offered the higher level of concentrates received a further 5 kg of T0 concentrate in two equal feeds per day through out-of-parlour feeders. Increasing the level of fish oil increased milk yield (P<0.01) and decreased the concentrations of fat (P<0.001) and protein (P<0.001). Increasing the level of fish oil decreased the concentrations of C6:0, C8:0, C10:0, C16:0, C14:0 and C18:0 and increased the concentrations of C18:1t, C20:0, C20:1, C20:4w6, C18:2c, C23:0, C20:5w3 and C18:2t. Relative to T300, T300B significantly decreased the concentrations of C20:4w6, but did not alter any of the other fatty acids. The transfer from feed to milk of EPA and DHA averaged 0.61 and 0.19 respectively across the three levels of fish oil supplementation. For T300 the efficiency of transfer of EPA was higher while the efficiency of transfer of DHA was lower to that for T300B. It is concluded that increasing fish oil supplementation decreased milk fat concentration by up to 15 g/kg. Also feeding fish oil is an efficient method of increasing EPA in the human diet through transfer into milk.