Effects on fatty acid profiles and milk fat yield due to dietary concentrate and supplemental 18:3n-3 were evaluated in 4 lactating Holstein cows fed a low- (35:65 concentrate:forage; L) or high- (65:35; H) concentrate diet without (LC, HC) added oil or with linseed oil (LCO, HCO) at 3% of DM. A 4 x 4 Latin square with four 4-wk periods was used. Milk yield and dry matter intake averaged 26.7 and 20.2 kg/d, respectively, across treatments. Plasma acetate and beta-hydroxybutyrate decreased, whereas glucose, nonesterified fatty acids, and leptin increased with high-concentrate diets. Milk fat percentage was lower in cows fed high-concentrate diets (2.31 vs. 3.38), resulting in decreases in yield of 11 (HC) and 42% (HCO). Reduced yields of 8:0-16:0 and cis9-18:1 fatty acids accounted for 69 and 17%, respectively, of the decrease in milk fat yield with HC vs. LC (-90 g/d), and for 26 and 33%, respectively, of the decrease with HCO vs. LCO (-400 g/d). Total trans-18:1 yield increased by 25 (HCO) and 59 (LCO) g/d with oil addition. Trans10-18:1 yield was 5-fold greater with high-concentrate diets. Trans11-18:1 increased by 13 (HCO) and 19 (LCO) g/d with oil addition. Trans13+14-18:1 yield increased by 9 (HCO) and 18 (LCO) g/d with linseed oil. Yield of total conjugated linoleic acids (CLA) in milk averaged 6 g/d with LC or HC compared with 14 g/d with LCO or HCO. Cis9,trans11-CLA yield was not affected by concentrate level but increased by 147% in response to oil. Feeding oil increased yields of trans11,cis13-, trans11,trans13-, and trans,trans-CLA, primarily with LCO. Trans10,cis12-CLA yield (average of 0.08 g/d) was not affected by treatments. Yield of trans11,cis15-18:2 was 1 g/d in cows fed LC or HC and 10 g/d with LCO or HCO. Yields of cis9,trans11-18:2, cis9,trans12-18:2, and cis9,trans13-18:2 were positively correlated (r = 0.74 to 0.94) with yields of trans11-18:1, trans12-18:1, and trans13+14-18:1, respectively. Plasma concentrations of biohydrogenation intermediates with concentrate or linseed oil level followed similar changes as those in milk fat. Milk fat depression was observed when HC induced an increase in trans10-18:1 yield. A correlation of 0.84 across 31 comparisons from 13 published studies, including the present one, was found among the increase in percentage of trans10-18:1 in milk fat and decreased milk fat yield. We observed, however, more drastic milk fat depression when HCO increased yields of total trans-18:1, trans11,cis15-18:2, trans isomers of 18:3, and reduced yields of 18:0 plus cis9-18:1.