SUMMARY: The control of dehydroepiandrosterone (DHA) synthesis in the human adrenal gland was studied by investigating the biosynthesis of its immediate precursor, 17-hydroxypregnenolone (3β,17α-dihydroxy-pregn-5-en-20-one) and the side-chain cleavage. Both conversions take place in the microsomal fraction and there is an obligatory requirement for the co-factor, reduced nicotinamide adenine dinucleotide phosphate (NADPH). Under optimal conditions, 17-hydroxylation is controlled only by the availability of the substrate and the co-factor. By using the same kinetic approach it was observed that the synthesis of DHA is controlled by the availability of substrate and co-factor. However, the reaction was inhibited by the oxidized form (NADP+) and when the ratio of NADP+:NADPH reached 1, the maximum velocity was halved. Two major metabolites of 17-hydroxypregnenolone, namely DHA and 17-hydroxyprogesterone, are non-competitive inhibitors of the reaction. The inhibition constants are of the magnitude of the tissue concentration of these steroids in the adrenal gland which suggests that non-competitive inhibition takes place in vivo.