Serum cortisol fractions were determined by isocolloidosmolar equilibrium dialysis in 93 women with breast cancer and 29 normal women. In breast cancer patients, the percentage of protein-unbound cortisol was increased at any concentration of total serum cortisol; this was accompanied by a relative decrease in the percentage of transcortin-bound cortisol. A significant increase in the unbound fraction is indicative of accelerated physiological activity of cortisol in breast cancer patients. In the breast cancer cases, there was a high incidence of obesity (61%) and impaired glucose tolerance (85%). In obese and/or diabetic patients, more remarkable increases of unbound cortisol were observed. We suggest that obesity and impaired glucose tolerance may increase the risk for breast cancer, and that the characteristic increase in the unbound cortisol fraction in women with breast cancer may reflect such risk factors.