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The purpose of this study was to determine if dietary factors could bias estimates of the relationships between estrogen metabolites and breast cancer risk factors. A lower ratio of urinary 2-hydroxyestrone/16alpha-hydroxyestrone (2/16) has been associated with breast cancer diagnosis. However, both estrogen metabolism and breast cancer risk have been associated with dietary intake, and breast cancer patients may have different dietary patterns than healthy controls. An association between urinary 2/16 levels and breast cancer risk may be due to transitory dietary change after diagnosis, or due to other breast cancer risk factors which have been associated to steroid hormone metabolism. Thirty-seven healthy postmenopausal women provided two 24-h urine samples at a two-week interval. Six 24-h diet recalls were administered in this same time period. In linear regression analysis, dietary fat-to-fiber ratio (fat/fiber) and the saturated fat/soluble fiber ratio was inversely associated with urinary 2/16 values (b = -0.22, 95% CI (-0.43, -0.01); b= -0.26, 95% CI (-0.43, -0.09), respectively). The effects of these dietary factors on 2/16 were independent of body mass index or other breast cancer risk factors. These study results suggest that some of the variation in estrogen metabolite levels among postmenopausal Caucasian women may be due to dietary intake, and that dietary factors should be carefully measured and evaluated when investigating the relationship between estrogen metabolites and breast cancer risk.