Pregnenolone sulphate (PREGS) is one of the most important neuroactive steroids. Previous study showed that PREGS enhanced long-term potentiation (LTP) via activation of post-synaptic NMDA receptors at excitatory synapses in the hippocampus. The present paper studied the effect of PREGS on LTP at excitatory synapses in the pyramidal cells of layers V-VI of the medial prefrontal cortex (mPFC) using whole-cell patch-clamp in slices and made a comparison with that in the hippocampus. We also studied the mechanism of the effect of PREGS in the mPFC. We found that PREGS inhibited induction of LTP in the mPFC and had no influence on NMDA currents, which was different from its effect in the hippocampus. Moreover, the effect of PREGS on LTP in the mPFC was cancelled by alpha2-adrenoreceptor antagonist, alpha2A-adrenoreceptor antagonist, Gi protein inhibitor, adenylate cyclase inhibitor and protein kinase A inhibitor. These results suggest that PREGS inhibits LTP via activation of the alpha2-adrenoreceptor-Gi protein-adenylate cyclase-protein kinase A signalling pathway in the mPFC.