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A simple method for the determination of cortisol-binding capacity is described. For saturation of the cortisol-binding proteins, plasma samples are incubated with an excess of cortisol. In the next step NADPH and liver microsomes of female rats are added. The microsomal Δ4-3-ketosteroid hydrogenase only reduces non protein-bound cortisol to tetrahydrocortisol-5α. Then the steroids are extracted by dichloromethane, and after some purification steps analyzed by fluorometry. Tetrahydrocortisol gives practically no fluorescence. The cortisol determined by this method corresponds to protein-bound cortisol and indicates the extent of cortisolbinding capacity. Precision and accuracy of the method were found to be good. The values of cortisol-binding capacity obtained by our method are compared with the results of other authors. The mean value of adult men was 25.5 ± 3.4 μg/100 ml, that of pregnant women, mens IX-X, 42.3 ± 4.2 μg/100 ml.