The results are reported of routine determinations of free C21-steroids in plasma and urine by a method which involved thin layer and gas liquid chromatography. The following steroids were simultaneously determined: Cortisol, cortisone, 6β-hydroxycortisol and -cortisone, 20-hydroxycortisol and -cortisone, tetrahydrocortisol and -cortisone, and 11-deoxycortisol (Reichstein S). The specificity, reproducibility and accuracy are discussed in detail. The overall recoveries of the procedure were examined by the addition of 1,2-3H-cortisol and 1,2-3H-cortisone prior to extraction. They ranged between 50 and 60 per cent. The method was clinically applied to various tests of adrenal cortical function and its pituitary regulation (e. g. ACTH test, dexamethasone suppression, metopirone test). The simultaneous determination in plasma and urine of a number of C21-steroid-hormones including their metabolites proved to yield valuable information about adrenal cortical function and C21-steroid metabolism. In the paediatric age group the metopirone test measuring 11-deoxycortisol in plasma rather than its tetrahydro derivative in urine was particularly useful since it avoided unpleasant 24 h-urine collections in young children.