Certain endogenously occurring 3α-hydroxylated, 5-reduced pregnane steroids act at a specific site on the GABAAreceptor complex (GRC) to modulate the effects of GABA at its receptor. Modulators that potentiate GABA at the GABAAreceptor often possess anxiolytic properties. The anxiolytic potential of four 5-reduced, 3α, 20-pregnanediols, differing only in the stereochemical orientation of the steroid A-ring and the 20-hydroxyl group, were tested in the Vogel test following intracerebroventricular (ICV) administration. The effects of these pregnanediols were compared to those of their 20-ketone analogues, 3α-hydroxy-5α-pregnan-20-one (3α,5α-P) and 3α-hydroxy-5β-pregnan-20-one (3α,5β-P). All four pregnanediols tested significantly enhanced punished drinking at doses ranging from 10 to 60 µg. The rank order of potency based on the minimum effective dose (MED) observed was 5α-pregnan-3α,20α-diol=5β-pregnan-3α,20α-diol>5β-pregnan-3α,20β-diol>5α-pregnan-3α,20β-diol. 3α,5β-P and 3α,5α-P enhanced punished responding when administered at 2.5 and 5 µg, respectively. 3β,5α-P which is inactive at the GRC was also inactive (up to 100 µg) in the Vogel test. The benzodiazepine control diazepam was efficacious when administered at 2.5 µg. 5α-Pregnan-3α,20α-diol was further tested in the mouse elevated plus-maze model following systemic administration where it was found to be active in a dose range of 10–40 mg/kg IP. These results raise the possibility that in addition to 3α,5α-P and 3α,5β-P, some of their endogenously occurring pregnanediol metabolites may also influence physiological