The epidemiology of breast cancer suggests a central etiologic role for premenopausal hormones, yet current data do not indicate which specific hormones are of greatest importance and at what levels risk is increased. We propose to analyze blood samples collected from the 29,613 participants in the Nurses' Health Study II (NHSII) who provided samples in 1996-99 and were 32 to 53 years of age at collection. 19,092 of these samples were timed according to the woman's menstrual cycle; for these women, both early follicular and midluteal blood samples are available. The samples have been stored at -130 degrees Centigrade or colder in liquid nitrogen freezers since collection. We will assay samples from women who were diagnosed with breast cancer after blood collection and matched controls who remained disease-free (i.e., a nested case-control design), thus efficiently utilizing these prospectively collected samples. Specifically, we will examine the relationships between breast cancer incidence and (1) plasma levels of estrogens, androgens and prolactin in the follicular and luteal phases of the menstrual cycle, (2) plasma levels of insulin-like growth factor I (IGF-I), IGF binding protein-3 (IGFBP-3), IGFBP-1 and free IGF-I, (3) polymorphisms in both the IGF-l and IGFBP-3 genes, (4) plasma levels of insulin and c-peptide, and (5) a polymorphism in the insulin receptor substrate I (IRS-i) gene. The ongoing NHSII cohort (CA50385) will provide follow-up of the cohort and documentation of breast cancer diagnoses in addition to providing information on important covariates (such as body mass index, parity, age at menarche, alcohol intake and family history of breast cancer) for the proposed study. Overall, the large size of the cohort, the prospective design, the high follow-up rate, the detailed exposure data, and the availability of archived blood specimens (with collection timed within the menstrual cycle) provide a unique opportunity to test several hypotheses of public health importance.