Lactating cows were fed menhaden fish oil to elevate concentrations of conjugated linoleic acid, transvaccenic acid, and n-3 fatty acids in milk. Twelve multiparous Holstein cows at 48+/-11 DIM were assigned randomly to a replicated 4 x 4 Latin square. Each treatment period was 35 d in length, with data collected d 15 to 35 of each period. On a dry matter (DM) basis, diets contained 25% corn silage, 25% alfalfa hay, and 50% of the respective concentrate mix. Fish oil was supplemented at 0, 1, 2, and 3% of ration DM. Linear decreases were observed for DM intake (28.8, 28.5, 23.4, and 20.4 kg/d) and milk fat (2.99, 2.79, 2.37, and 2.30%) for 0 to 3% dietary fish oil, respectively. Milk yield (31.7, 34.2, 32.3, and 27.4 kg/d) increased as dietary fish oil increased from 0 to 1% but decreased linearly from 1 to 3% dietary fish oil. Milk protein percentages (3.17, 3.19, 3.21, and 3.17) were similar for all treatments. When the 2% fish oil diet was fed, concentrations of conjugated linoleic acid and transvaccenic acid in milk fat increased to 356% (to 2.2 g/ 100 g of total fatty acids) and 502% (to 6.1 g/100 g), respectively, of amounts when 0% fish oil was fed. There were no additional increases in these fatty acids when cows were fed 3% fish oil. The n-3 fatty acids increased from a trace to over 1 g/100 g of milk fatty acids, when the 3% fish oil diet was fed. Fish oil supplementation to diets of dairy cows increased the conjugated linoleic acid, transvaccenic acid, and n-3 fatty acids in milk.