A multitude of different serotonin (5-HT) receptor types are expressed in the hippocampus, but the identity of receptors actually mediating the physiological response to endogenous 5-HT has not been determined. We combined pharmacologically induced release of 5-HT with patch-clamp recordings on disinhibited rat CA1 minislices to determine effects of endogenous 5-HT on the excitability of pyramidal neurons and synaptic transmission among them. We found that application of 5-HT releasers, 3,4-methylenedioxy-methamphetamine (MDMA) or p-methylthioamphetamine, at concentrations ranging from 2 to 50 microm, reduced the excitatory synaptic transmission between CA1 pyramidal neurons without altering their basal electrical properties. This effect of MDMA was blocked by the selective 5-HT1B antagonist GR 55562, was dependent on endogenous 5-HT content and was mediated by presynaptically located, pertussis-toxin sensitive mechanisms. We found no other MDMA effects in our preparation, which indicates that the release of endogenous 5-HT preferentially stimulates 5-HT1B receptors on CA1 pyramidal neurons. Therefore, 5-HT1B receptor activation may represent a predominant component of the physiological response to endogenous 5-HT in the CA1. The high sensitivity of the 5-HT1B receptor-mediated reduction of polysynaptic excitatory responses to the extracellular 5-HT level enabled us to study mechanisms of the 5-HT releasing action of MDMA. Block of the serotonin transporter (SERT) with citalopram slowed the time course and reduced overall 5-HT release by MDMA. Depletion of vesicular 5-HT, by inhibition of vesicular monoamine transporter type 2 with tetrabenazine prevented the release. Thus although the SERT reversal contributes, a direct vesicle-depleting action is essential for MDMA release of 5-HT.