Accurate determination of the flow of nutrients at the omasum requires the use of a triple marker system. Typically, a system based on ruminal administration of the lithium salt of CoEDTA, ytterbium acetate (Yb-Ac), and chromium-mordanted straw (Cr-S) has been used. However, there is evidence to suggest that product:substrate ratios for stearoyl-coenzyme A desaturase (Delta(9)-desaturase) are lower in milk fat from cows administered a combination of CoEDTA, Yb-Ac, and Cr-S, indicating reduced Delta(9)-desaturase activity. To evaluate this hypothesis, samples of milk were collected 1 d before, and on d 2, 6, and 9 of administering the CoEDTA, Yb-Ac, and Cr-S triple marker system into the rumen of 4 cows. A 4 x 4 Latin square with 28-d experimental periods was used to assess the effects of 0, 75, 150, and 300 g/d of fish oil in the diet on ruminal and mammary lipid metabolism. Irrespective of the amount of fish oil in the diet, concentrations of all milk fatty acids containing a cis-9 double bond were reduced after markers were given. Milk fatty acid pairs dependent on Delta(9)-desaturase were decreased over time, with responses reaching a nadir within 6 d of marker administration. Overall, administering markers into the rumen was associated with a mean decrease in milk cis-9 10:1/ 10:0, cis-9 12:1/12:0, cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 17:1/17:0, cis-9 18:1/18:0, and cis-9,trans-11 conjugated linoleic acid/trans-11 18:1 concentration ratios of 44.6, 52.7, 58.7, 36.8, 37.2, 44.3, and 43.0%, respectively. In conclusion, one or more of the markers administered altered milk fatty acid composition and may act as an inhibitor of Delta(9)-desaturase in the bovine mammary gland.