There is some evidence that glucose and other factors related to glucose metabolism, such as insulin and insulin-like growth-factors (IGFs) may contribute to breast cancer development. The present study analyzed the hypothesis that serum glucose, insulin levels, and IGF-I pattern are associated with breast cancer using a nested case-control study. Between 1987 and 1992, 10,786 women ages 35-69 were recruited in a prospective study in Italy. Women with history of cancer and on hormone therapy were excluded at baseline. At recruitment, blood samples were collected after a 12-h fast between 7:30 and 9:00 a.m. from all of the study participants. After 5.5 years, 144 breast cancer cases were identified among the participants of the cohort. Four matched controls were chosen for each breast cancer case from members of the cohort who did not develop breast cancer during the follow-up period. In premenopausal women, glucose was associated with breast cancer risk: the age, body mass index, and reproductive variable adjusted relative risk (RR) for the highest quartile of serum glucose versus the lowest was 2.8 [95% confidence interval (CI), 1.2-6.5], and P for trend was 0.02. Insulin showed a weaker association with breast cancer, the adjusted RR of the highest quartile versus the lowest was 1.7 (95% CI, 0.7-4.1), and P for trend was 0.14, whereas the adjusted RR of the highest quartile of IGF-I was 3.1 (95% CI, 1.1-8.6), and P for trend was 0.01. Increased levels of insulin-like growth factor binding protein-3 (IGFBP)-3 were related to breast cancer risk: the adjusted RR for the highest quartile was 2.1 (95% CI, 0.95-4.75), and P for trend was 0.02. In postmenopausal women, the associations of glucose, insulin, and IGF-1 pattern were associated with breast cancer risk in heavier subjects characterized by a body mass index higher than 26. These results indicate that chronic alteration of glucose metabolism is related to breast cancer development.