By using custom multielectrode probes suitable for hippocampal neural circuits, we have investigated rapid regulation of the long-term potentiation (LTP) by neurosteroids in rat hippocampal slices. Neurosteroids include pregnenolone, estradiol and testosterone which are shown to be synthesized from cholesterol within the hippocampus in addition to testis and ovary. We blocked the synthesis of pregnenolone, by inhibiting the activity of its synthetic enzyme, cytochrome P450scc, with an inhibitor AG. AG perfusion for 20 min significantly suppressed LTP. AG perfusion also rapidly decreased EPSP (excitatory postsynaptic potentiation). We can rescue the suppression of LTP and EPSP by supplementation of exogenous pregnenolone. These results show that the effect of pregnenolone to rapidly regulate neuronal synaptic plasticity. We also searched for pregnenolone receptor that mediates its action in the brain. We analyze a pregnenolone receptor, CLIP-170. We found that pregnenolone binds to and changes the conformation of CLIP-170 into an active form. When activated by pregnenolone, CLIP-170 could bind to microtubules and increased microtubule polymerization. Since pregnenolone supplementation therapy has been applied to rescue spinal cord injuries, our analysis could be useful for understanding its molecular mechanisms.