The associations between serum concentrations of oestradiol, testosterone and sex hormone-binding globulin (SHBG) and risk of breast cancer in post-menopausal women were investigated in a prospective study on the island of Guernsey. Sixty-one women who developed breast cancer an average of 7.8 years after blood collection were matched for age, year of blood collection and number of years post-menopausal with 179 control subjects. Women using exogenous hormones at the time of blood collection were excluded from the study. Women who subsequently developed breast cancer had a 29% higher geometric mean oestradiol concentration than control women (P = 0.004). The odds ratio for breast cancer in the top third compared with the lowest third of the oestradiol concentration distribution was 5.03 (95% confidence interval 2.02-12.49, P for trend < 0.001). Adjusting for testosterone and SHBG concentrations did not substantially alter the odds ratio for oestradiol. Although testosterone and SHBG concentrations were associated with breast cancer risk, the concentrations of these hormones were correlated with those of oestradiol; the associations were not statistically significant after adjusting for oestradiol concentration. These data provide evidence that serum oestradiol concentrations in post-menopausal women may have a substantial effect on breast cancer risk.