Immortalized hypothalamic GT1-7 neurons represent a good model system to investigate the control of gonadotropin-releasing hormone (GnRH) secretion. Using these cells, we observed that the neuroactive steroid pregnenolone sulfate (PREGS) is able to stimulate the release of GnRH in a dose-dependent manner through N-Methyl-D-Aspartate (NMDA) receptors, since its action is completely blocked by a specific NMDA receptor antagonist and magnesium. GT1-7 neurons express mRNAs for different mouse NMDA receptor subunits (,1, 3, 4 and 2, corresponding to the NR1, NR2C, NR2D, and NR2B rat subunits), and increase their spontaneous release of GnRH, when incubated in the presence of exogenous glutamate or NMDA. In addition, we found that these neurons are able to release and synthesize glutamate, as demonstrated by the presence of glutamate accumulated in the defined incubation medium of the neurons, along the time of the experiment and the expression of the mRNA coding for the glutamate transporter VGLUT2, a specific marker of glutamatergic neurons. The potentiating effect of PREGS on the secretion of GnRH induced by glutamate is consistent with the role of the steroid as a positive allosteric modulator of NMDA receptors. Alltogether, these results point to a novel mechanism by which the neuroactive steroid PREGS may potentiate an autocrine excitatory loop in GnRH neurons.