OBJECTIVE Two isoforms of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) catalyse the interconversion of cortisol to hormonally inactive cortisone; defects in the 11β‐HSD2 isoform result in hypertension. The kidney, expressing high levels of 11β‐HSD2, is the principal source of cortisone in man. We have validated the measurement of urinary free cortisone (UFE) excretion in normals and in patients with disorders of the pituitary‐adrenal axis in an attempt to more accurately measure the activity of 11β‐HSD2in vivo.SUBJECTS Forty‐one normal adults, 12 normal children<12 years of age, 15 patients with Cushing’s syndrome, 12 with hypopituitarism on replacement hydrocortisone, 12 with the syndrome of apparent mineralocorticoid excess (AME) and 7 volunteers consuming liquorice.MEASUREMENTS A complete 24‐hour urine collection was analysed by gas chromatography/mass spectrometry for ‘A‐ring’ reduced cortisol and cortisone metabolites, i.e. tetrahydrocortisols (THF and allo‐THF) and tetrahydrocortisone (THE). In addition, urinary free cortisol (UFF) and urinary free cortisone were quantified using deuterium‐labelled internal standards.RESULTS In normal adults and children, UFE excretion exceeded that of UFF (UFF 30.4 ± 2.4 μg/24h (mean ± SE), UFE 54.6 ±4.1 μg/24h, adults) (for conversion to nmol/24h multiply E by 2.78 and F by 2.76 respectively). Thus the normal UFF/UFE ratio was 0.54 ± 0.05 in contrast to the (THF+allo‐THF)/THE ratio of 1.21 ± 0.06. UFE excretion was normal in hypopituitary patients on replacement hydrocortisone. Although UFE was elevated in all forms of Cushing’s syndrome, the UFF/UFE ratio was grossly elevated in patients with the ectopic ACTH syndrome (14.0 ± 6.7,n=6). UFE was below the lower limit of the assay (<1 μg/24h) in most patients with the so‐called type 1 variant of AME and significantly reduced in 4 patients described as having the type 2 variant of AME (10.5 ± 3.5 μg/h,P<0.05) and in 7 volunteers consuming liquorice (26.8 ± 10.0 μg/24h,P<0.01). In ectopic ACTH syndrome, AME, and liquorice ingestion the UFF/UFE ratio was more deranged than the (THF+ allo‐THF)/THE ratio.CONCLUSION In normals the discrepant THF + allo‐THF/THE and UFF/UFE ratio suggests that much more of the UFE is derived from the kidney. Reduction in UFE excretion is seen following liquorice ingestion and in both variants of AME, though it is more profound in AME1. The high UFF/UFE ratio in the mineralocorticoid excess state seen in the ectopic ACTH syndrome is compatible with substrate‐saturation of renal 11β‐HSD2. The measurement of UFE and the UFF/UFE ratio is a significant advance in the analysis of human 11β‐HSD activityin vivo; in particular,