This study investigated the mode of action of conantokin-T, a 21 amino acid peptide toxin isolated from the venom of the fish-hunting cone snail Conus tulipa, on excitatory synaptic transmission in rat hippocampal slices using intracellular recording techniques. Superfusion of conantokin-T (1-500 nM) specifically and irreversibly decreased the pharmacologically isolated N-methyl-D-aspartate receptor (NMDA)-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner but had no effect on normal excitatory synaptic transmission (EPSP). The sensitivity of postsynaptic neurons to NMDA but not to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid was also antagonized by conantokin-T pretreatment. In addition, the conantokin-T-induced depression of EPSPNMDA could be antagonized by prior treatment of hippocampal slices with either DL-2-amino-5-phosphonovaleate (10 microM) or ifenprodil (20 microM). However, 7-chlorokynurenic acid (1 microM) had no effect on the action of conantokin-T. These findings indicated that conantokin-T modulates the NMDA receptor by an interaction with its glutamate binding site and polyamine recognition site.