Background: It is well accepted that exercise can decrease breast cancer risk. Limited clinical evidence suggests that this risk could be mediated through changes in estrogen metabolism in premenopausal women. Our objective was to investigate the effects of exercise on premenopausal estrogen metabolism pertinent to breast cancer risk. Methods: Sedentary, healthy, young eumenorrheic women were randomized into an intervention of 30 minutes of moderate-to-vigorous aerobic exercise five times a week for approximately 16 weeks (n = 212), or into a usual-lifestyle sedentary control group (n = 179). Urinary levels of estrogens [estrone [E1], estradiol, and estriol] and nine estrogen metabolites were measured at baseline and at study end by liquid chromatography/ tandem mass spectrometry. The ratios of 2-hydroxyestrone to 16a-hydroxyestrone (2-OHE1/16a-OHE1) and 2- OHE 1 to 4-hydroxyestrone (2- OHE1/4-OHE1) were also calculated. Results: The exercise intervention resulted in significant increases in aerobic fitness and lean body mass and a significant decrease in percent body fat. For exerciserswhocompleted the study (n=165),2-OHE 1/16a-OHE1 increased significantly (P = 0.043), whereas E1 decreased significantly (P = 0.030) in control participants (n = 153). The change from baseline in 2-OHE1/16alpha;-OHE1 was significantly different between groups (P = 0.045), even after adjustment for baseline values. Conclusions: The exercise intervention resulted in a significant increase in the 2-OHE1/16alpha;-OHE1 ratio but no differences in other estrogen metabolites or ratios. Impact: Our results suggest that changes in premenopausal estrogen metabolism may be a mechanism by which increased physical activity lowers breast cancer risk. Cancer Epidemiol Biomarkers Prev; 22(5); 756-64.