There is considerable scientific interest in whether measurement of the major estrogen metabolites 2- and 16 alpha-hydroxyestrone will shed light on the role of estrogen in the risk of breast cancer. These have been difficult to measure in large numbers because of the need for radiolabeled tracers, but a new assay is able to utilize spot urine samples. The main objective of this study was to assess the reliability of a newly developed enzyme immunoassay (EIA) for the measurement of 2- and 16 alpha-hydroxyestrone in urine samples collected from a large group of healthy premenopausal women enrolled in a clinical trial A secondary objective was to assess the impact of several factors such as body weight on the urinary estrogen metabolite ratios. The study cohort included 174 women aged 44-50, who were enrolled in the Cardiovascular Risk Factors and Menopause Trial, also referred to as the Women's Healthy Lifestyle Project (WHLP), an ongoing 5-year clinical trial of 535 premenopausal women randomized either to an intensive dietary life-style intervention group or to an assessment-only control group. Measurements of 2- and 16 alpha-hydroxyestrone showed a high intraclass correlation for blind duplicate urine samples (R = 0.94 and R = 0.80), cross-sectionally and over time (R = 0.79 and R = 0.62), in this population of healthy premenopausal women. The intervention diet (of 25% of total calories from fat) did not appear to influence the estrogen metabolite ratio. This new estrogen metabolite EIA demonstrates good reliability and thus may be appropriate for use in large epidemiologic studies of estrogen-related diseases. There was no relation between dietary fat reduction, weight loss, and increased exercise and change in the ratio among premenopausal women in this study.