The conditions for optimal yields from human urine of corticosteroids and 17-ketosteroids were investigated by means of different methods of hydrolysis and extraction. With β-glucuronidase hydrolysis and simultaneous dialysis, almost all tetrahydro derivatives of cortisone, hydrocortisone, corticosterone, as well as etiocholanolone, androsterone and 11-oxygenated 17-ketosteroids are liberated. The dialysis does not proceed quantitatively, particularly with regard to the isolation of 17-ketosteroids. Hydrocortisone and cortisone seem to occur as free steroids in human urine. They can be extracted at pH 7.0 without preceding hydrolysis. The continuous extraction with ether or ethyl acetate at pH 1.0 hydrolyses the sulphuric acid esters of dehydroepiandrosterone and androsterone, but no measurable amounts of corticosteroid conjugates. Extraction at an acid pH leads to considerable spoiling of the extracts by unspecific tetrazolium blue positive chromogens. For the simultaneous hydrolysis and extraction of corticosteroids and 17-ketosteroids from one urine sample the following procedure is proposed: β Glucuronidase hydrolysis and dialysis for three days; extraction at pH 7.0 with ether of the watery contents of the dialyzing tube; continuous extraction with ether at pH 0.5 for 48 hours.