GABA(A) receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3alpha,5[alpha]beta-THP (3alphaOH-5[alpha]beta-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by alpha1beta2delta GABAR, we have shown that 3alpha,5[alpha]beta-THP effects at recombinant alpha4beta2delta GABAR depend on the direction of Cl(-) flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of alpha4beta2delta GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3alpha,5[alpha]beta-THP was not observed at other receptor subtypes, suggesting it was selective for alpha4beta2delta GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of alpha4betadelta GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3alpha,5[alpha]beta-THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl(-) gradient. By decreasing this shunting inhibition, 3alpha,5[alpha]beta-THP increased the excitability of CA1 hippocampal pyramidal cells at puberty. These effects of the steroid were opposite to those observed before puberty when 3alpha,5[alpha]beta-THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3alpha,5[alpha]beta-THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of alpha4beta2delta GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence.