We examined the effect of intake of fresh pasture on concentrations of conjugated linoleic acid in milk fat. Sixteen Holstein cows were paired and divided into either the control group or the grazing group. The study involved initial, transition, and final periods. During the initial period, all cows consumed a total mixed diet. Cows in the control group were fed the total mixed diet throughout the study, and cows in the grazing group were gradually adjusted to a diet consisting of intensively managed pasture. Performance of cows in the grazing group was significantly reduced from that of cows in the control group during the final period (dry matter intake, 19% less; milk yield, 29.6 vs. 44.1 kg/d; and live weight, 40 kg less). During the initial period, when both groups were consuming a total mixed diet, concentrations of conjugated linoleic acid in milk fat were similar (X = 5.1 mg/g of milk fat). As the grazing group was gradually adjusted to pasture, concentrations of conjugated linoleic acid in milk gradually increased. During the final period, when cows in the grazing group were consuming a diet consisting of pasture only, conjugated linoleic acid concentrations in the milk fat were doubled (10.9 vs. 4.6 mg/g of milk fat). Furthermore, results showed the individual consistency of the milk fat content of conjugated linoleic acid over time but also demonstrated substantial variation among individual cows within treatment groups. Overall, this study indicated that the concentration of conjugated linoleic acid in milk fat is enhanced by dietary intake of fresh pasture.