NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) action at NMDA receptors. 3alpha5betaS was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3alpha5betaS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3alpha5betaS in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3alpha5betaS at NMDA receptors. In accordance with the model, 3alpha5betaS was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3alpha5betaS is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.