The objective of this experiment was to evaluate the effect of forage source on the temporal changes in milk conjugated linoleic acid (CLA). Fourteen Holstein cows were divided into two groups. One group (LOT) was fed a corn silage-alfalfa hay mix ad libitum, while the other group (PAS) grazed on alfalfa-grass pasture. Additionally, both groups were fed 8.2 kg d-1 grain supplement containing 650 g of oil composed of fish oil and sunflower oil (1:4). The concentration of cis-9, trans-11 CLA in milk fat of both diets reached maximum levels on day 3 of oil supplementation, declined during day 6, and then remained constant during the reminder of the experiment. Milk vaccenic acid (VA) and trans-10 C18:1 reached their maximum concentrations on days 3 and 6, respectively, of oil supplementation with both diets. In conclusion, the decline in milk cis-9, trans-11 CLA over time was not affected by forage source and may have resulted from an increase in formation of trans-10 C18:1 and C18:0 at the expenses of VA. Key words: Conjugated linoleic acid, milk, forage, fish oil, sunflower oil