Dehydroepiandrosterone sulfate (DHEAS) is one of the most important neuroactive steroids. The present study examined the effect of DHEAS on spontaneous and evoked glutamate release in the pyramidal cells of layers V and VI of the rat prelimbic cortex by using whole-cell patch-clamp recordings in slices and further investigated its mechanism. The results showed that DHEAS at 1 microM had no effect on spontaneous glutamate release but inhibited 5-hydroxytryptaime (5-HT)-evoked glutamate release. The concentration-response relationship of this effect of DHEAS was U-shaped with a maximum at 1 microM, and this inhibition seemed to have some extent of selectivity for the 5-HT-evoked glutamate release because it had no effects on high K(+)-, electrical stimulus-, and dopamine-evoked releases. Further study showed that DHEAS inhibited the 5-HT(3) receptor agonist evoked-glutamate release but had no effect on the 5-HT(2A/2C) receptor agonist-evoked release. Moreover, the 5-HT(3) receptor antagonist could block the effect of DHEAS on the 5-HT-evoked glutamate release. The mechanism study showed that the sigma-1 receptor antagonist could block the effect of DHEAS and that the sigma-1 receptor agonist could mimic the effect of DHEAS on 5-HT(3) receptor agonist-evoked glutamate release and intrasynaptosomal Ca(2+) increase. These results suggest that DHEAS can inhibit 5-HT-evoked glutamate release via activation of the sigma-1 receptor and then inhibition of the 5-HT(3) receptor in the pyramidal cells of the prelimbic cortex.