In the assay of corticosteroids in plasma and urine by competitive protein binding, the use of horse serum or plasma as a source of assay protein gives better sensitivity and somewhat better specificity for cortisol than do previously described procedures. Five to 10 µl of plasma unknown or 50 to 100 µl of urine unknown can be used. Precision for the standard curve is 0.12 ng in the range 0-2.00 ng, and accuracy, measured as the amount of added cortisol recovered from plasma, is about 91%. Because of the improved specificity, the need for preliminary purification by chromatography is decreased for some purposes. The same corticosteroid-binding globulin and standard curve can be used for assaying corticosteroids in urine or plasma. The procedure may be useful for unusually small samples or if discriminations within the lower range of physiological concentrations are needed, as in work with infants and neonates or in the study of small adrenal secretory pulses, such as those occurring at the nadir of the circadian cycle.