Olanzapine increases brain allopregnanolone (ALLO) levels sufficiently to modulate neuronal activity by allosterically regulating GABAA receptors. Recently, we reported the antipsychotic-like profile of ALLO in rodents. The present study examined the hypothesis that olanzapine-induced elevation of endogenous neurosteroid ALLO is vital for its neuroleptic-like action. The conditioned avoidance response (CAR) and apomorphine-induced climbing behavioral paradigms were used in rodents. Administration of ALLO (1 microg, intracerebroventricular (i.c.v.)) or neurosteroidogenic agents such as the mitochondrial diazepam binding inhibitor receptor agonist, FGIN 1-27 (0.5 microg, i.c.v.) or the ALLO precursor, progesterone (10 mg/kg, i.p.) significantly potentiated olanzapine-induced blockade of CAR and apomorphine-induced climbing. In contrast, these agents failed to alter the antipsychotic-like effect of risperidone and haloperidol. On the other hand, inhibition of the endogenous biosynthesis of neurosteroids by the 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg, i.p.), the 3alpha-hydroxysteroid oxidoreductase inhibitor, indomethacin (5 mg/kg, i.p.), or the GABAA receptor antagonist bicuculline (1 mg/kg, i.p.) and dehydroepiandrosterone sulfate (DHEAS) (1 mg/kg, i.p.) blocked the effect of olanzapine, but not of risperidone and haloperidol. Socially isolated animals, known to exhibit decreased brain ALLO and GABAA receptor functions, displayed a shortening in the muscimol-induced loss of righting reflex and an increased susceptibility to apomorphine-induced climbing. Administration of olanzapine, but not of haloperidol and risperidone, normalized the duration of muscimol-elicited loss of righting reflex. Although all three antipsychotics proved capable of antagonizing the apomorphine-induced climbing, a dose almost five times higher of olanzapine was required in socially isolated animals. The data obtained suggest that enhancement of the GABAergic tone plays a key role in the antipsychotic-like effect exerted by olanzapine in rodents, likely as a consequence of augmented levels of neuroactive steroids, in particular ALLO, in the brain. The present findings provide the first specific behavioral evidence in support of the hypothesis that neuroactive steroid ALLO- mediated GABAergic modulation is essential for the antipsychotic-like action of olanzapine.