The isomerization of the Δ5-3-ketosteroid isoprogesterone into the Δ4-3-ketosteroid progesterone has been examined with recombinant 3β-hydroxysteroid dehydrogenase from Digitalis lanata (rDl3βHSD), partially purified 3-ketosteroid isomerase from Digitalis lanata (Dl3KSI) and under non-enzymatic conditions in deuterium oxide (D2O).Studies indicate that the isomerization catalyzed by the Dl3KSI proceeds without significant isotope exchange between the medium and the steroid and thus involves an intramolecular proton transfer consistent with the mechanism of the bacterial 3-ketosteroid isomerase of Pseudomonas testosteroni. For the rDl3βHSD as well as under non-enzymatic conditions deuterium was incorporated from the incubation buffer during isomerization. Together with a comparison of the rate of isomerization under the different conditions, it was demonstrated that rDl3βHSD does not possess 3-ketosteroid isomerase activity.