SUMMARY A method is described for determining the degree of binding by plasma proteins of cortisol by a technique of pressure ultrafiltration at 37° after addition of radioactively labelled cortisol. In normal subjects, the mean concentration of transcortin binding sites was 5·82 × 10−7 moles/l. and the apparent association constant at 37° was 4·27 × 107 l./mole. Evidence of increased protein binding of cortisol was obtained in oestrogen-treated subjects and in women in the last 3 months of pregnancy. In the latter subjects normal levels of non-protein bound cortisol were found. The mean level of non-protein bound cortisol in eight men treated with stilboestrol was increased, but normal levels were present in four of these subjects. The protein binding of cortisol in three patients with Cushing's syndrome and in three patients with Addison's disease was normal. Thirty-five% of cortisol in adrenal venous plasma was in the unbound state. Increased levels of plasma 17-hydroxycorticosteroids (17-OHCS) were found in patients in stressful situations (post-operative states, infections and burns). There was no evidence of increased protein binding of cortisol. Patients at death had increased levels of plasma 17-OHCS including three patients who had been on long-term steroid therapy. There was no evidence of increased protein binding of cortisol. On the contrary, there was some evidence of a decrease in transcortin binding sites. It is concluded that stressful situations in man, regardless of the outcome, are associated with increased plasma levels of biologically active cortisol.