Conantokin-G is a 17 amino acid peptide isolated from the venom of the fish-eating snail Conus geographus which produces hyperactivity when injected into the brains of adult mice. We show that this peptide is a selective N-methyl-D-aspartate (NMDA) antagonist based on its ability to block NMDA-induced elevation of cGMP in rat cerebellar slices in vitro (IC50 = 171 nM), but not kainic acid-induced elevations. This inhibition could not be overcome by increasing the NMDA concentration, indicating non-competitive inhibition. Conantokin-G displayed no affinity for binding sites for thienylcyclohexylpiperidine, various glutamate subclasses or those for several other neurotransmitters/neuromodulators. This peptide, however, enhanced [3H]glycine binding to rat forebrain membranes but not to spinal cord membranes. The activity profile of the peptide in various assays indicates that it is a novel type of non-competitive NMDA antagonist.