Dynorphin A (DynA), an endogenous agonist of kappa-opioid receptors, has also been reported to directly interact with the NMDA receptor. DynA inhibition of NMDA receptor function has been suggested to be involved in its neuroprotective action during ischemic and acidic conditions. However, the effect of external pH on DynA inhibition of the NMDA receptor has not been reported. Here, we show that DynA inhibition of the NMDA receptor is dependent on extracellular pH over the range of pH 6.7-8.3, and the inhibition by 10 microM DynA increases at low pH by three- to four-fold in hippocampal neurons and in Xenopus oocytes expressing NR1-1a/2B subunits. Molecular studies showed that the interacting site for DynA on the NMDA receptor is distinct from that of proton or redox sites. Peptide mapping demonstrated important contributions of positively charged residues and specific structural organization of the peptide to the potency of DynA inhibition. Thus, DynA inhibits NMDA receptors through an allosteric mechanism, which is pH dependent and involves the specific structural features of the peptide.