A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 micrograms/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.