We have developed a new assay for cortisone (E) in serum, saliva, and urine involving Celite® chromatography followed by RIA with 125I-labeled E and scintillation proximity assay. The chromatography step separates cortisol (F) from E, and in combination with their RIAs, permits assessment of the status of the F–E shuttle. We report the results of basal, postcorticotropin (ACTH), and postdexamethasone E and F concentrations and their circadian fluctuations in the serum, saliva, and urine of healthy volunteers. The serum and urine F/E ratios were increased in patients with ectopic ACTH secretion, whereas in adrenal adenoma and Cushing disease only the urinary ratio was increased. In chronic renal insufficiency this ratio was increased in serum (23.5 ± 3.9) but diminished in saliva (0.38 ± 0.11), and in apparent mineralocorticoid excess the ratios were high in serum (44.3 ± 9.3) and urine (5.35 ± 0.85) compared with those of healthy subjects (serum 9.8 ± 3.5, urine 0.52 ± 0.29, saliva 0.52 ± 0.29).