Previous prospective studies have raised the possibility that the antioxidantproperties of carotenoids and vitamin E (alpha-tocopherol) and the role of vitamin A (retinol) in cellular differentiation may be associated with a reduced risk of subsequent breast cancer. To investigate the association between serum and plasma concentrations of retinol, retinyl palmitate, alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, total-carotenoids, alpha-tocopherol, and gamma-tocopherol with subsequent development of breast cancer, a nested case control study was conducted among female residents of Washington County, Maryland, who had donated blood for a serum bank in 1974 or 1989. Cases (n = 295) and controls (n = 295) were matched on age, race, menopausal status, and date of blood donation, and the analyses were stratified by cohort participation. Median concentrations of beta-carotene, lycopene, and total carotene were significantly lower in cases compared with controls in the 1974 cohort (13.1, 12.5, and 7.9% difference; P = 0.01, 0.04, and 0.04, respectively) and for lutein in the 1989 cohort (6.7% difference; P = 0.02). The risk of developing breast cancer in the highest fifth was approximately half of that of women in the lowest fifth for beta-carotene [odds ratio (OR) = 0.41; 95% confidence interval (CI) 0.22-0.79; P trend = 0.007], lycopene (OR = 0.55; 95% CI 0.29-1.06; P trend = 0.04), and total carotene (OR = 0.55; 95% CI 0.29-1.03; P trend = 0.02) in the 1974 cohort. There was generally a protective association for other micronutrients in both cohorts, although none reached statistical significance. The results suggest that carotenoids may protect against the development of breast cancer.