Due to its potential effects on ovarian hormone production, physical activity has been proposed as a modifiable risk factor for breast cancer. The authors analyzed data from the American Cancer Society Cancer Prevention Study II (CPS-II) Nutrition Cohort to examine the association between various measures of physical activity and postmenopausal breast cancer risk. Information on physical activity was obtained in 1992 via a self-administered questionnaire for 72,608 postmenopausal female participants who were cancer-free. During the five year prospective follow-up, 1520 incident breast cancer cases were identified among these women. Cox proportional hazards modeling was used to compute hazard rate ratios (RR) and to adjust for potential confounding factors including mammography. Women who were most physically active (> 42.0 MET-h/week) at baseline had 29% lower incidence rates than active women with the least activity (> 0-7.0 MET-h/week) (95% CI, 0.49-1.02). The difference in risk was largest for localized breast cancer, and for women who did not use hormone replacement therapy (HRT) at enrollment. Our findings are consistent with other studies that show lower risk of postmenopausal breast cancer associated with regular physical activity.