Conjugated octadecadienoic acids (18:2, conjugated linoleic acids) have been shown to be anticarcinogenic and may influence growth and nutrient partitioning. The delta 9c,11t-18:2 isomer (rumenic acid, RA) is most common in both food sources and human tissues. To determine if maternal diet can influence milk RA concentration, breastfeeding women (n = 16) were enrolled in a 3-wk crossover study. Women initially consumed minimal amounts of food containing RA during week 1, then were assigned randomly to consume diets rich in high-fat dairy foods (and thus RA) during week 2 or 3. Milk was collected by complete breast expression twice during each experimental week. Current and chronic RA intakes were estimated by 3-d dietary records and food frequency questionnaires, respectively. Estimated chronic RA intakes ranged from 49 to 659 mg/d. Dietary RA intake was greater during the high compared to the low dairy period (291 +/- 75 vs. 15 +/- 24 mg/d, respectively; P < 0.0001). Milk contained more RA during the high than the low dairy period (13.5 +/- 0.1 vs. 8.2 +/- 0.4 mumol/g lipid, respectively; P < 0.0001). Milk lipid concentration was influenced by diet, such that lipid concentration was greater during the high than the low dairy period (46.6 +/- 5.0 vs. 38.3 +/- 1.6 mg/g milk, respectively; P < 0.05). Additionally, multiple regression analyses suggested that body mass index was the primary predictor of milk RA and lipid concentrations. In summary, these data indicate that both lipid and RA concentrations of human milk can be influenced by diet.