Loss-of-function mutations or inhibition of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2) results in overstimulation of the mineralocorticoid receptor by cortisol and causes salt-sensitive hypertension. Traditionally, 11beta-HSD-2 activity has been assessed by measurement of the urinary cortisol metabolite ratio (tetrahydrocortisol [THF]+5alpha-THF)/tetrahydrocortisone (THE). Recently, the ratio of urinary free glucocorticoids, UFF/UFE, has been suggested to be a more reliable parameter, an aspect that has not been investigated systematically. Steroid metabolites were measured repeatedly by gas chromatography-mass spectrometry in 20 healthy subjects at baseline and after 1 week each of a 30- or 180-mmol/d of sodium diet or 500 mg/d of glycyrrhetinic acid. Intraindividual coefficients of variation from 3 random urine collections for (THF+5alpha-THF)/THE and UFF/UFE ratios were 11+/-9% and 25+/-14% (P<0.001). (THF+5alpha-THF)/THE was more sensitive than UFF/UFE for detection of glycyrrhetinic acid-induced increases higher than the upper 95% confidence interval of the coefficient of variation of the corresponding ratio. Low- or high-salt diet did not alter either ratio. Mean (THF+5alpha-THF)/THE but not UFF/UFE was higher in salt-sensitive than salt-resistant subjects. Absolute glycyrrhetinic acid-related increase in (THF+5alpha-THF)/THE but not UFF/UFE was higher in salt-sensitive than salt-resistant subjects and correlated with changes in mean BP. Intraindividual variability of (THF+5alpha-THF)/THE is lower than that of UFF/UFE. The UFF/UFE ratio does not appear to be more sensitive than (THF+5alpha-THF)/THE for detection of decreased 11beta-HSD-2 activity. The (THF+5alpha-THF)/THE ratio better discriminates between salt-sensitive and salt-resistant subjects. Together with BP responses to glycyrrhetinic acid, these findings support a pivotal role of 11beta-HSD-2 in salt sensitivity.