A method is described for the quant. determination of 20-oxo steroids in urine that uses 3α,20β-hydroxysteroid:NAD-oxidoreductase (EC 1.1.1.53). In the presence of NADH, the enzyme catalyzes the reduction of 20-oxo groups of the C21 steroids to 20β-alcs. in an equimolar reaction. The consumption of NADH is a measure for the steroid concentrations present. The concentration of tetrahydrocorticosterone was determined 45 min after the start of the reaction. The method gives good results with regard to accuracy and precision, but the specificity for tetrahydrocorticosterone remains to be proven.