SUMMARY: The Δ5-3β-hydroxysteroid dehydrogenase activity of an acetone powder of bovine adrenal microsomes has been studied using 3β-hydroxyandrost-5-en-17-one and 3β-hydroxypregn-5-en-20-one as substrates. The products were separated, identified and quantitated by gas—liquid chromatographymass spectrometry. The Michaelis constants for both steroid substrates and for NAD have been derived and are of the order of 10−6 m. Although kinetic studies indicate the existence of a single Δ5-3β-hydroxysteroid dehydrogenase, the possibility of a non-enzymic rate-limiting step common to both steroid substrates and to NAD must be entertained.