Using a 4-step scheme, we have synthesized deuterium-labelled 17α-hydroxypregnenolone for use as internal standard for an isotope dilution/mass spectrometry plasma assay. 17α-Hydroxyprogesterone served as starting material. Its 17α-hydroxygroup was protected by tetrahydropyranylation. Introduction of deuterium by base-catalyzed enolization and protection of the 3-hydroxygroup by acetate formation yielded [2,2,4,21,21,21-2H6]3β,17α-dihydroxypregna-3,5-diene-20-one 3-acetate 17-tetrahydro-pyranyl ether. After reductive deuteration of the 3-ene and removal of the protecting groups [2,2,4,4,21,21,21-2H7] 17α-hydroxypregnenolone was obtained. © 1998 John Wiley & Sons, Ltd.