The Δ5-3β-hydroxysteroid dehydrogenase activity of a 10 000 × g × 10 min supernatant of porcine ovarian tissue homogenate has been studied using pregnenolone and dehydroepiandrosterone as substrates. The products, progesterone and androstenedione respectively, were isolated by repeated paper and thin layer chromatography in a radiochemically pure form, and the reaction velocities were calculated from the amount of product isolated. The apparent Michaelis-Menten's constants were 2.6 × 10−7 mol/l for pregnenolone and 1.1 × 10−7 mol/l for dehydroepiandrosterone. These low values indicate that the enzyme has very strong affinity for the substrate. This may possibly be the basis for the inability of the porcine ovary to produce androstenedione in vitro via the Δ5-pathway. At a pregnenolone concentration of 4.8 × 10−6 mol/l a significant substrate inhibition was observed, but no such inhibition was seen with dehydroepiandrosterone. A synthetic gestagen, chlormadinone acetate, inhibited 3β-hydroxysteroid dehydrogenase activity. The inhibition was non-competitive with regard to the pregnenolone conversion, and of a more complex type for dehydroepiandrosterone. At equimolar concentrations of substrate and inhibitor, 1.5 × 10−6 mol/, the inhibition was of the order of 80-85%. This inhibition may be quite important in the ovary of the sow because of the inability of this tissue to utilize the Δ5-pathwaw for androstenedione formation.