Based on the potential benefits to human health, there is interest in developing sustainable nutritional strategies to enhance the concentration of long-chain n-3 fatty acids in ruminant-derived foods. Four Aberdeen Angus steers fitted with rumen and duodenal cannulae were used in a 4 x 4 Latin square experiment with 21 d experimental periods to examine the potential of fish oil (FO) in the diet to enhance the supply of 20 : 5n-3 and 22 : 6n-3 available for absorption in growing cattle. Treatments consisted of total mixed rations based on maize silage fed at a rate of 85 g DM/kg live weight 0.75/d containing 0, 8, 16 and 24 g FO/kg diet DM. Supplements of FO reduced linearly (P < 0.01) DM intake and shifted (P < 0.01) rumen fermentation towards propionate at the expense of acetate and butyrate. FO in the diet enhanced linearly (P < 0.05) the flow of trans-16 : 1, trans-18 : 1, trans-18 : 2, 20 : 5n-3 and 22 : 6n-3, and decreased linearly (P < 0.05) 18 : 0 and 18 : 3n-3 at the duodenum. Increases in the flow of trans-18 : 1 were isomer dependent and were determined primarily by higher amounts of trans-11 reaching the duodenum. In conclusion, FO alters ruminal lipid metabolism of growing cattle in a dose-dependent manner consistent with an inhibition of ruminal biohydrogenation, and enhances the amount of long-chain n-3 fatty acids at the duodenum, but the increases are marginal due to extensive biohydrogenation in the rumen.