Lycopene is potentially effective in the prevention of breast cancer from laboratory and observational studies. Among 39,876 women initially free of cardiovascular disease and cancer, we first conducted a prospective cohort study of dietary lycopene and its food sources. Participants completed a baseline food frequency questionnaire and provided self-reports of breast cancer risk factors. Dietary lycopene levels were divided into quintiles, and lycopene food sources were categorized. During 9.9 years of follow-up, 1,076 breast cancer cases were confirmed by medical record review. In a nested case-control study, we then identified 508 breast cancer cases and 508 controls matched by age, smoking, and follow-up time. Plasma lycopene and other carotenoids were measured. In the prospective cohort study, women with increasing quintiles of dietary lycopene had multivariate relative risks (RR) of breast cancer of 1.00 (ref), 0.95, 1.00, 1.10, and 1.00 (P, linear trend = 0.71). Women consuming <1.5, 1.5 to <4, 4 to <7, 7 to <10, and > or =10 servings/week of tomato-based products had RRs of 1.00 (ref), 1.00, 1.20, 1.18, and 1.16 (P, linear trend = 0.11). No individual lycopene food sources were associated with breast cancer. In the nested case-control study, women in increasing quartiles of plasma lycopene had multivariate RRs of breast cancer of 1.00 (ref), 0.95, 1.15, and 0.93 (P, linear trend = 0.86). The stepwise addition of individual plasma carotenoids did not impact the RRs for plasma lycopene, nor were other carotenoids associated with breast cancer. In conclusion, neither higher dietary nor plasma lycopene levels were associated with a reduced risk of breast cancer in middle-aged and older women.