Abstract Rat hypothalamic 5α-dihydroprogesterone NADH-linked 3α-hydroxysteroid oxidoreductase, (3α-HSD) activity, which is associated with plasma membranes, has a relatively sharp pH optimum of 5.5 and a temperature optimum range of 45–52°C. This enzyme exhibited apparent K m 's for the reductive reaction of 0.40 ± 0.09 μm and 29 ± 12 μ M for 5α-dihydroprogesterone and NADH, respectively. For the oxidative reaction, apparent K m 's of 0.11 ± 0.01 μ M and 84.3 ± 22.8 μ M were observed for 3α-hydroxy-5α-pregnan-20-one and NAD + respectively. The NADPH-linked 3α-HSD activity, which is present in the cytosol, proceeded optimally between pH 6–10 and at a temperature of 45°C. This enzyme exhibited apparent K m 's for the reductive reaction of 0.083 ± 0.009 μ M and 0.71 ± 0.10 μ M for 5α-dihydroprogesterone and NADPH, respectively. For the oxidative reaction, apparent K m 's of 2.33 ± 0.15 μ M and 21.0 ± 3.1 μ M were observed for 3α-hydroxy-5α-pregnan-20-one and NADP + , respectively. The results suggest that the NADH-linked and NADPH-linked 5α-dihydroprogesterone 3α-HSD activities of female rat hypothalamus differ in their intra-cellular interconversion of 5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one.