Glutamate is recognized as a prominent excitatory transmitter in the supraoptic nucleus (SON) and is involved in transmission of osmoregulatory information from the osmoreceptors to the vasopressin (VP) and oxytocin (OT) neurons. Explants of the hypothalamo-neurohypophysial system were utilized to characterize the roles of the non-N-methyl-D-aspartate (NMDA) glutamate receptor subtypes (non-NMDA-Rs), kainic acid receptors (KA-Rs), and aminopropionic acid receptors (AMPA-Rs) and to evaluate the interdependence of NMDA-Rs and non-NMDA-Rs in eliciting hormone release. Although both KA and AMPA increased hormone release, a specific agonist of the KA-Rs, SYM-2081, was not effective. This combined with the finding that cyclothiazide, an agent that inhibits the desensitization of AMPA-Rs, increased the VP response to both KA and AMPA indicates that the increase in hormone release induced by the non-NMDA agonists is mediated via AMPA-Rs, rather than KA-Rs. Inhibition of osmotically stimulated VP and OT release by a specific AMPA-R antagonist indicated that AMPA-Rs are essential for mediating osmotically stimulated hormone release. NMDA-stimulated VP but not OT release was prevented by blockade of non-NMDA-Rs, but AMPA-stimulated VP/OT release was not prevented by NMDA-R blockade.