Previous studies suggest that the estrogen metabolite 16alpha-hydroxyestrone acts as a breast tumor promoter. The alternative product of estrogen metabolism, 2-hydroxyestrone, does not exhibit estrogenic properties in breast tissue, and lower values of the ratio 2-hydroxyestrone:16alpha-hydroxyestrone (2:16) in urine may be an endocrine biomarker for greater breast cancer risk. Vegetables of the Brassica genus, such as broccoli, contain a phytochemical, which may shift estrogen metabolism and increase the 2:16 ratio. Adding 500 g/day of broccoli to a standard diet shifts 2:16 values upward in humans; however, it is unknown as to whether healthy women are able to consume a sufficient quantity of Brassica to affect breast cancer risk through this mechanism. In this study, 34 healthy postmenopausal women participated in an intensive intervention designed to facilitate the addition of Brassica to the daily diet. The diet was measured by repeated 24-h recall, and estrogen metabolites were measured by enzyme immunoassay in 24-h urine samples. In a crude analysis, there was a nonsignificant increase in the urinary 2:16 ratio associated with greater Brassica consumption. With adjustment for other dietary parameters, Brassica vegetable consumption was associated with a statistically significant increase in 2:16 values, such that for each 10-g/day increase in Brassica consumption, there was an increase in the 2:16 ratio of 0.08 (95% confidence interval, 0.02-0.15). To the extent that the 2:16 ratio, as measured in urine, is associated with breast cancer risk, future research should consider Brassica vegetable consumption as a potentially effective and acceptable dietary strategy to prevent breast cancer.