Huperzine A (HUP-A), first isolated from the Chinese club moss Huperzia serrata, is a potent, reversible and selective inhibitor of acetylcholinesterase (AChE) over butyrylcholinesterase (BChE) (Life Sci. 54: 991-997). Because HUP-A has been shown to penetrate the blood-brain barrier, is more stable than the carbamates used as pretreatments for organophosphate poisoning (OP) and the HUP-A:AChE complex has a longer half-life than other prophylactic sequestering agents, HUP-A has been proposed as a pretreatment drug for nerve agent toxicity by protecting AChE from irreversible OP-induced phosphonylation. More recently (NeuroReport 8: 963-968), pretreatment of embryonic neuronal cultures with HUP-A reduced glutamate-induced cell death and also decreased glutamate-induced calcium mobilization. These results suggest that HUP-A might interfere with and be beneficial for excitatory amino acid overstimulation, such as seen in ischemia, where persistent elevation of internal calcium levels by activation of the N-methyl-D-aspartate (NMDA) glutamate subtype receptor is found. We have now investigated the interaction of HUP-A with glutamate receptors. Freshly frozen cortex or synaptic plasma membranes were used, providing 60-90% specific radioligand binding. Huperzine A (< or =100 microM) had no effect on the binding of [3H]glutamate (low- and high-affinity glutamate sites), [3H]MDL 105,519 (NMDA glycine regulatory site), [3H]ifenprodil (NMDA polyamine site) or [3H]CGS 19755 (NMDA antagonist). In contrast with these results, HUP-A non-competitively (Hill slope < 1) inhibited [3H]MK-801 and [3H]TCP binding (co-located NMDA ion channel PCP site) with pseudo K(i) approximately 6 microM. Furthermore, when neuronal cultures were pretreated with HUP-A for 45 min prior to NMDA exposure, HUP-A dose-dependently inhibited the NMDA-induced toxicity. Although HUP-A has been implicated to interact with cholinergic receptors, it was without effect at 100 microM on muscarinic (measured by inhibition of [3H]QNB or [3H]NMS binding) or nicotinic [3H]epibatidine binding) receptors; also, HUP-A did not perturb adenosine receptor binding [3H]PIA or [3H]NECA). Therefore, HUP-A most likely attenuates excitatory amino acid toxicity by blocking the NMDA ion channel and subsequent Ca2+ mobilization at or near the PCP and MK-801 ligand sites. Thus, on the one hand, HUP-A could be used as a pretreatment against OPs and it might also be a valuable therapeutic intervention in a variety of acute and chronic disorders by protecting against overstimulation of the excitatory amino acid pathway. By blocking NMDA ion channels without psychotomimetic side-effects, HUP-A may protect against diverse neurodegenerative states observed during ischemia or Alzheimer's disease.