Physical activity has been found to be associated with decreased risk of breast cancer in postmenopausal women in the majority of epidemiologic studies, but the association is inconsistent in premenopausal women. We studied the effect of physical activity at various ages on the incidence of breast cancer in 99,504 women from 30 to 49 years of age at enrollment in the Women's Lifestyle and Health Study, a prospective population-based cohort study in Norway and Sweden. Physical activity level on an ordinal scale at age 14, age 30, and age at enrollment, participation in competitive sports, as well as information on other covariates was obtained using a self-administered questionnaire. Complete follow-up with data on incident invasive breast cancer and mortality was collected by linkage to national registries. The relation between physical activity and time to breast cancer development was assessed using Cox proportional hazard models, controlling for potential confounders. During an average 9.1 years of follow-up, there were 1,166 incident breast cancer cases. The mean age of the women was 41 years at enrollment, and the mean age at breast cancer diagnosis was 48 years. Compared to inactive women, women with higher levels of physical activity at enrollment had a similar risk of incident breast cancer (adjusted relative risk, 1.24 for vigorous activity compared with no activity; 95% CI, 0.85-1.82). Physical activity at age 30 or at age 14 also did not afford any significant protection from breast cancer, nor did a consistently high level of activity from younger ages to enrollment. We did not find evidence of a protective effect of physical activity on breast cancer risk in this group of primarily premenopausal women.