Calcium has anti-proliferative and pro-differentiation effects on mammary cells in vitro and can inhibit the development of mammary tumors in mice. While there is some epidemiologic evidence for an inverse relation between dietary calcium intake and breast cancer risk, only one previous study has examined serum calcium levels in relation to breast cancer risk. We investigated this relation in a prospective cohort study of 2,762 women, aged 43-86, who were enrolled in the Beaver Dam Eye Study in 1988 and followed for up to 19 years. We found no evidence for an association between breast cancer risk and either total (hazard ratio, HR = 0.98; 95% CI: 0.60-1.60; 4(th) vs. 1(st) quartile) or ionized (HR = 0.85; 95% CI: 0.53, 1.38; 4(th) vs. 1(st) quartile) serum calcium levels. Additionally, we found no evidence for an association among subgroups defined by menopausal status and body mass index.