Abstract The relationship between unbound and total cortisol has been studied in patients with a variety of clinical conditions. We report on a simple, rapid and reliable method for evaluating the percentage unbound cortisol in serum or plasma which can be used in conjunction with total plasma cortisol to obtain a more complete understanding of the patients' adrenal status. Comparison of a patient's percentage unbound and total cortisol with a nomogram showing the normal relationship between percentage unbound and total cortisol indicates the patient's concentration of cortisol binding globulin. The concentration of unbound cortisol, the biologically active moiety, is the product of the percentage unbound and total cortisol concentration. The following values for the unbound cortisol concentration (μg/dl) were obtained (mean ± S.D.). Twenty-four normal subjects, 8–10 a.m. 1.2 ± 0.4; 14 women receiving an oral contraceptive 1.4 ± 0.5; 6 patients with adrenal insufficiency 0.2 ± 0.1; 9 hyperthyroid patients 1.7 ± 0.8; 5 acute ill patients 3.8 ± 2.4; and a patient with Cushing's syndrome 6.1. In normal subjects the values decreased at 4 p.m. (0.4 ± 0.1) and after dexamethasone administration (0.1 ± 0.1), and increased following the intravenous injection of adrenocorticotropic hormone (3.6 ± 0.7). In pregnant women the unbound cortisol increased as the pregnancy progressed, first trimester: 1.2 ± 0.3, second trimester: 1.6 ± 0.2, third trimester: 2.4 ± 0.5.