Rumen biohydrogenation of dietary polyunsaturated fatty acids (PUFA) is a significant limitation on any attempt to manipulate the PUFA content of ruminant products (meat or milk). This study examined rumen biohydrogenation of PUFA, the effects of PUFA on other aspects of rumen metabolism and fatty acid flow to and digestion in the small intestine of steers fed on different sources of lipid. Animals were fed ad libitum on grass silage and one of four concentrates (60:40 forage:concentrate on a dry matter basis) containing differing sources of lipid: megalac (rich in C16:0; M), linseed (rich in C18:3n-3; L), fish oil (rich in C20:5n-3 and C22:6n-3; FO) and a mixture of linseed/fish oil (LFO). Diets were formulated so that total dietary oil intake was approximately 60 g/kg of the DM intake, approximately half of which was from the experimental test oil. Rumen NH3-N (P = 0·09) and total VFA concentrations (P = 0·007) were higher on L, FO and LFO compared to M. Dry matter intakes did not differ across treatments and averaged 7·2 kg/day. Intake and flow of fatty acids to the duodenum was 323, 438, 344 and 381 (S.E.M. 9·1; P < 0·001) and 432, 489, 412 and 465 (S.E.M. 18·5; P < 0·1) g/day for M, L, FO and LFO, respectively. Biohydrogenation of C18:1n-9 was lower than all the other unsaturated fatty acids and it was lower of FO and LFO compared to M and L, on average 66·1 and 72·2 %, respectively. Biohydrogenation of C18:2n-6 averaged 89·8 % across treatments and was lower (P < 0·05) on M compared to L and FO. Biohydrogenation of C18:3n-3 averaged 92·1 % across treatments and was lowest on M (88·8 %) and highest on L and LFO (94·3 %). Biohydrogenation of C20:5n-3 and C22:6n-3 averaged approximately 91 and 89 % across the treatments, respectively. Small intestinal digestibilities of all fatty acids were high. In conclusion, feeding different sources of lipid with different fatty acid composition had significant effects on rumen function. The PUFA in whole linseed were only partially protected from biohydrogenation by the seed coat and in contrast to previous reports the C20 PUFA in fish oil were biohydrogenated to a large extent.