3 alpha-hydroxylated pregnane steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, metabolites of progesterone and deoxycorticosterone, 5 alpha-pregnan-3 alpha-o1-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha,21-diol-20-one (5 alpha-THDOC), respectively, were tested for anxiolytic effects in N.I.H. Swiss-Webster mice using the light/dark transition, open-field and lick-suppression tests. Similar to the benzodiazepine (BZ) diazepam, 3 alpha-OH-DHP (5-40 mg/kg) and 5 alpha-THDOC (5-40 mg/kg) significantly increased the number of light/dark transitions. 3 alpha-OH-DHP's effects were stereospecific as its diasteriomer, 3 beta-OH-DHP was devoid of activity. The benzodiazepine antagonist CGS-8216 (10 mg/kg) blocked diazepam's (1.0 mg/kg) anxiolytic effects, but did not have any effect against 3 alpha-OH-DHP (20 mg/kg). The data indicate that the pregnane steroids produce their anxiolytic effects through a separate mechanism than the BZs. 3 alpha-OH-DHP (20 mg/kg), 5 alpha-THDOC (20 mg/kg) and diazepam (1.0 mg/kg) increased activity in a open-field test. 3 beta-OH-DHP had no effect in the open-field test. Furthermore, 3 alpha-OH-DHP produced a 235% increase in punished responding in a lick-suppression test. These results demonstrate that the endogenous pregnane steroids possess anxiolytic effects that may be clinically relevant.