A control diet and a fish oil diet were fed to 12 multiparous Holstein cows to determine how the incorporation of Menhaden fish oil in the diet would influence the fatty acid composition, especially the conjugated linoleic acid and transvaccenic acid, contents of milk and butter. The control diet consisted of a 50:50 ratio of forage to concentrate, and the fish oil diet consisted of the control diet with 2% (on a dry matter basis) added fish oil. Milk from cows fed the control diet contained higher average concentrations of milk fat (3.37%) compared with milk from cows fed the fish oil diet (2.29%). Milk from cows fed fish oil contained higher concentrations of conjugated linoleic acid, transvaccenic acid, and total unsaturated fatty acids (0.68 and 2.51; 1.42 and 6.28; and 30.47 and 41.71 g/100 g of fat, respectively). Butter made from the fish oil diet milk also had higher concentrations of conjugated linoleic acid, transvaccenic acid, and unsaturated fatty acids. Penetrometer readings indicated fish oil diet butters were softer at 4 and 20 degrees C than the control diet butters. Acid degree values were similar in the fish oil butters compared with the control butters. No significant difference was found in the flavor characteristics of milk and butter from cows fed the control and fish oil diets. Production of milk and butter with increased amounts of conjugated linoleic acid, transvaccenic acid, and other beneficial fatty acids may have a desirable impact on the health of consumers and lead to increased sales.