Pregnenolone sulfate, one of the most abundantly produced neurosteroids in the hippocampus, has well characterized effects at postsynaptic receptors including the N-methyl-D-asparate type of glutamate receptor. Little is known, however, about the mechanism of action of neurosteroids on the release of glutamate. In this study we describe a robust effect of pregnenolone sulfate at glutamatergic synapses in the CA1 region of the hippocampus. In particular, we found that pregnenolone sulfate enhances paired-pulse facilitation of EPSPs at the two major classes of ionotropic glutamate receptors with an EC(50)<1 microM. Thus, we propose a novel mechanism of action of neurosteroids in hippocampal neurons that involves the modulation of glutamate release.