This study examined the effects of neurosteroids on the behavior of mice in the mirrored chamber test of anxiety, and determined the potential mechanisms by which neurosteroids alter the behavior in animal models of anxiety. Allopregnanolone (AP) (0.5 and 1 mg/kg) and pregnenolone sulfate (PS) (0.5 and 2 mg/kg) significantly reduced the latency to enter the chamber, and increased both number of entries and total time spent in the chamber in a dose-dependent manner, without affecting the spontaneous locomotor activity. In contrast, dehydroepiandrosterone sulfate (DHEAS) (1 and 2 mg/kg) increased motor activity and caused an anxiogenic response, i.e., an increase in latency to enter the mirrored chamber, and a decrease in the number of entries and time spent in the chamber. Progesterone (PROG) (1-10 mg/kg), a neurosteroid precursor, and 4'-chlordiazepam (4'-CD) (0.25-1 mg/kg), a specific ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor (MDR), produced a clear dose-dependent anxiolytic response in the mirrored chamber. The AP-, PROG- and 4'-CD-elicited anxiolytic behavior was blocked by picrotoxin (1 mg/kg), a GABA-A chloride channel antagonist, but not by flumazenil (2 mg/kg), a selective benzodiazepine (BZD) antagonist. In contrast, the anxiolytic effect of PS was not blocked by picrotoxin. The 4'-CD-induced anxiolytic effect was prevented by pretreatment with PK11195 (2 mg/kg), a selective partial MDR antagonist. Nifedipine (2 and 5 mg/kg), a dihydropyridine-type Ca2+ channel blocker, produced a flumazenil-resistant anxiolytic effect. Combined administration of nifedipine (2 and 5 mg/kg) and PS (0.5 and 2 mg/kg) exerted a significant additive effect in the mirrored chamber test. The potent anxiolytic effect of dizocilpine (0.5 and 1 mg/kg), an NMDA receptor antagonist, was blocked by pretreatment with DHEAS (2 mg/kg). Neurosteroids evoked changes in mirrored chamber activities resembling those elicited by triazolam (0.25 and 0.5 mg/kg). However, these effects were seen at doses that did not markedly affect locomotor activity, thereby suggesting these changes in behavior represent anxiolytic actions. Together, these results provide evidence for differential behavioral actions of the neurosteroids AP, PS and DHEAS in the mirrored chamber test of anxiety. The anxiolytic effect of PROG may be imputed to its metabolism to neurosteroid AP, while the 4'-CD-induced anxiolytic response is related to its MDR-stimulated neurosteroidogenesis and subsequent modulation of GABA-A receptor. Further, these differential effects reaffirm the contention that neurosteroids could be involved in the homeostasis of stress response.