A method proposed by Kitabchi and Kitchell [Anal. Biochem. 34, 529 (1970)] for the fluorometric determination of plasma 11-hydroxycorticosteroids has been modified. It is simplified by eliminating centrifugations, by processing all samples consecutively (rather than in small groups), by using disposable test tubes, and by prealkalinizing standards and blanks as well as plasma samples. Specificity and sensitivity are increased by measuring fluorescence at 520 nm, with an excitation wavelength of 470 nm. Effects of prealkalinization and time of fluorescence development on final cortisol values were studied. Large fluorescence increases are possible after 60 min of fluorescence development. Cortisol recoveries were not changed by the use of phase-separating filter paper nor were cortisol values altered by partial aging of the fluorescence reagent. Sensitivity, specificity, accuracy, and precision of the proposed method are reported.