The objective was to evaluate different levels of sun-flower oil (SFO) in dairy rations to increase vaccenic (trans-11-18:1) and rumenic acids (cis-9,trans-11-18:2) in milk fat, and assess the content and composition of other trans-octadecenoic (trans-18:1) and conjugated linoleic acids (CLA) isomers. Eighty lactating Holstein cows were fed control diets for 4 wk and then placed on 4 diets for 38 d; milk fat was analyzed after 10 and 38 d. The treatments were: control, 1.5% SFO plus 0.5% fish oil (FO), 3% SFO plus 0.5% FO, and 4.5% SFO plus 0.5% FO. The forage-to-concentrate ratio was 50:50 and consisted of barley/alfalfa/hay silage and corn/barley grain concentrate. There were no differences in milk production. Supplementation of SFO/FO reduced milk fat compared with respective pretreatment periods, but milk protein and lactose levels were not affected. There was a linear decrease in all short- and medium-chain saturated fatty acids (SFA) in milk fat after 10 d (25.5, 24.1, 20.2, and 16.7%) and a corresponding linear increase in total trans-18:1 (5.2, 9.1, 14.1, and 21.3%) and total CLA (0.7, 1.9, 2.4, and 3.9%). The other FA in milk fat were not affected. Separation of trans-18:1 isomers was achieved by combination of gas chromatography (GC; 100-m highly polar capillary column) and prior separation of trans FA by silver ion-thin layer chromatography followed by GC. The CLA isomers were resolved by a combination of GC and silver ion-HPLC. The trans-11- and trans-10-18:1 isomers accounted for ∼50% of the total trans-18:1 increase when SFO/FO diets were fed. On continued feeding to 38 d, trans-11-18:1 increased with 1.5% SFO/FO, stayed the same with 3%, and declined with 4.5% SFO/FO. Rumenic acid showed a similar pattern on continued feeding as trans-11-18:2; levels increased to 0.43, 1.5, 1.9, and 3.4% at 10 d and to 0.42, 2.15, 2.09, and 2.78% at 38 d. Rumenic acid was the major CLA isomer in all 4 diets: 66, 77, 78 and 85%. The CLA isomers trans-7,cis-9-, trans-9,cis-11-, trans-10,cis-12-, trans-11,trans-13-, and trans-9,trans-11-/trans-10,trans-12-18:2 also increased from 0.18 (control) to 0.52% (4.5% SFO/FO). Milk fat produced from 3% SFO/FO appeared most promising: trans-11-18:1 and cis-9,trans-11-18:2 increased 4.5-fold, total SFA reduced 18%, and moderate levels of trans-10-18:1 (3.2%), other trans-18:1 (6.6%) and CLA isomers (0.5%) were observed, and that composition remained unchanged to 38 d. The 4.5% SFO/FO diet produced higher levels of trans-11-18:1 and cis-9,trans-11-18:2, a 28% reduction in SFA, and similar levels of other trans-18:1 (9.2%) and CLA isomers (0.52%), but the higher levels of trans-11-18:1 and cis-9,trans-11-18:2 were not sustained. A stable milk fat quality was achieved by feeding moderate amounts of SFO (3% of DM) in the presence of 0.5% FO that had 4% vaccenic and 2% rumenic acids.