Given the possible breast cancer preventive effects of isoflavones, substances with an estrogen-like structure occurring in soybeans, it is of interest to assess levels directly in the breast. The objective of this pilot study was to measure isoflavones in nipple aspirate fluid. In a pilot study with 11 women, nipple aspirate fluid and urine samples were collected at baseline and after 1 month of consuming two servings of soymilk per day. In a parallel investigation, one woman donated nipple aspirate fluid, blood, and urine on the same day after consuming different amounts of soy for 9 days. Nipple aspirate fluid was collected with an aspirator, diluted 1 : 11, and stored at -20°C. Isoflavonoids in nipple aspirate fluid, serum, and urine were liquid-liquid extracted after enzymatic hydrolysis followed by liquid chromatography electrospray ionization high resolution tandem mass spectrometry analysis using triply 13C labeled internal isoflavonoid standards. After the 30-day soy intervention, median nipple aspirate fluid yield changed very little (P=0.47), whereas urinary isoflavonoid excretion (P=0.04) and isoflavonoid levels in nipple aspirate fluid (P=0.12) increased substantially. For the nine samples collected in one participant, isoflavonoids measured in nipple aspirate fluid, plasma, and urine were highly correlated with the number of daily soy servings and with each other. This pilot study indicates that isoflavonoids in nipple aspirate fluid are 10 times lower than in plasma and are closely related to levels of isoflavonoids in urine and plasma. These findings show that isoflavonoids are present in breast fluid and may act directly on breast tissue.