Enhancing the polyunsaturated fatty acid (PUFA) and decreasing the saturated fatty acid content of beef is an important target in terms of improving the nutritional value of this food for the consumer. The present study examined the effects of feeding a ruminally protected lipid supplement (PLS) rich in PUFA on the fatty acid composition of longissimus thoracis muscle and associated subcutaneous adipose tissue. Animals were fed ad libitum on grass silage plus one of three concentrate treatments in which the lipid source was either Megalac (rich in palmitic acid; 16:0) or PLS (soyabean, linseed and sunflower-seed oils resulting in an 18:2n−6:18:3n−3 value of 2·4:1). Treatment 1 contained 100g Megalac/kg (Mega, control); treatment 2 (PLS1) contained 54g Megalac/kg with 500g PLS/d fed separately; treatment 3 (PLS2) contained no Megalac and 1000g PLS/d fed separately. The PLS was considered as part of the overall concentrate allocation per d in maintaining an overall forage:concentrate value of 60:40 on a DM basis. Total dietary fat was formulated to be 0·07 of DM of which 0·04 was the test oil. Total intramuscular fatty acids (mg/100g muscle) were decreased by 0·31 when feeding PLS2 compared with Mega (P<0·05). In neutral lipid, the PLS increased the proportion of 18:2n−6 and 18:3n−3 by 2·7 and 4·1 on diets PLS1 and PLS2 v. Mega, respectively. Similar responses were noted for these fatty acids in phospholipid. The amounts or proportions of 20:4n−6, 20:5n−3 or 22:6n−3 were not influenced by diet whereas the amounts and proportions of 22:4n−6 and 22:5n−3 in phospholipid were decreased with inclusion of the PLS. The amounts of the saturated fatty acids, 14:0, 16:0 and 18:0, in neutral lipid were on average 0·37 lower on treatment PLS2 compared with Mega. Feeding the PLS also decreased the proportion of 16:0 in neutral lipid. The amount of 18:1n-9 (P=0·1) and the amount and proportion of 18:1 trans (P<0·01) were lower on treatments PLS1 and PLS2 in neutral lipid and phospholipid. Conjugated linoleic acid (cis-9, trans-11) was not influenced by diet in the major storage fraction for this fatty acid, neutral lipid. The PUFA:saturated fatty acids value was increased markedly (×2·5) with inclusion of the PLS (P<0·001) while the σn−6:n−3 value increased slightly (×1·2; P=0·015). The results suggest that the protected lipid used, which was rich in PUFA, had a high degree of protection from the hydrogenating action of rumen micro-organisms. The PLS resulted in meat with a lower content of total fat, decreased saturated fatty acids and much higher 18:2n−6 and 18:3n−3. The net result was a large shift in polyunsaturated: saturated fatty acids, 0·28 v. 0·08, on feeding PLS2 compared with Mega, respectively.