We describe the first method of routine measurement of prednisone, prednisolone and 6 beta-hydroxyprednisolone concomitantly in urine. Urine (3 ml) is extracted with ethyl acetate, washed with base and separated by high-performance liquid chromatography on a silica column with a solvent system of hexane--diethyl ether--ethanol--tetrahydrofuran--glacial acetic acid (59.9:31:2.3:6.5:0.3, v/v). The steroids are detected at 254 nm. Because no conventional internal standard was found, 6 beta-[3H]hydroxycortisol and [3H]prednisolone are added to urine prior to extraction; 3H is monitored by a radioactivity detector coupled with the chromatograph. The assay exhibits linearity from 200 to 7500 ng and an inter-day variability of less than 11.4% (C.V.).