In a previous paper a method was described by which sulphuric esters and glucuronides of steroids could be separated by paper electrophoresis and chromatography. This method was applied to the separation and quantitative determination of the 17-ketosteroids present in urinary extracts after they had been converted to the corresponding sulphuric esters. The present paper deals with the application of the above-mentioned technique to the separation and quantitative determination of the conjugated 17-ketosteroids, directly extracted from urine by means of n -butanol. A sample of 200 ml of 24-hour urine is sufficient for the determination; the conjugated 17-ketosteroids are separated from the pigments and fractionated in two zones corresponding to sulphates and glucuronides, respectively.