Body mass index (BMI) is inversely related to the risk of premenopausal breast cancer, but the underlying biological mechanisms of this association are poorly understood. Leptin, a peptide hormone produced primarily by adipocytes, is a potential mediator of the BMI association because BMI and total body fat are positively associated with circulating leptin levels and leptin and its receptor are overexpressed in breast tumors. We conducted a prospective case-control study nested within the Nurses' Health Study II cohort examining the association between plasma leptin levels in premenopausal women and breast cancer risk. Leptin was measured in blood samples collected between 1996 and 1999. The analysis included 330 incident breast cancer cases diagnosed after blood collection and 636 matched controls. Logistic regression models, controlling for breast cancer risk factors, were used to calculate ORs and 95% CIs. After adjustment for BMI at age 18, weight change since age 18 to blood draw, and other breast cancer risk factors, plasma leptin levels were inversely associated with breast cancer risk (OR for top vs. bottom quartile = 0.55; 95% CI = 0.31-0.99; P(trend) = 0.04). Adjustment for BMI at blood draw attenuated the association (OR = 0.69; 95% CI = 0.38-1.23; P(trend) = 0.26). Our results suggest that leptin may be inversely associated with breast cancer risk, but it is unclear whether any part of this association is independent of BMI.