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NMDA receptor (NMDAR) antagonists are dissociative anesthetics, drugs of abuse, and are of therapeutic interest in neurodegeneration and neuropsychiatric disease. Many well-known NMDAR antagonists are positively charged, voltage-dependent channel blockers. We recently showed that the hydrophobic anion dipicrylamine (DPA) negatively regulates GABA(A) receptor function by a mechanism indistinguishable from that of sulfated neurosteroids. Because sulfated neurosteroids also modulate NMDARs, here we examined the effects of DPA on NMDAR function. In rat hippocampal neurons DPA inhibited currents gated by 300 µM NMDA with an IC(50) of 2.3 µM. Neither onset nor offset of antagonism exhibited dependence on channel activation but exhibited a noncompetitive profile. DPA antagonism was independent of NMDAR subunit composition and was similar at extrasynaptic and total receptor populations. Surprisingly, similar to cationic channel blockers but unlike sulfated neurosteroids, DPA antagonism was voltage dependent. Onset and offset of DPA antagonism were nearly 10-fold faster than DPA-induced increases in membrane capacitance, suggesting that membrane interactions do not directly explain antagonism. Furthermore, voltage dependence did not derive from association of DPA with a site on NMDARs directly accessible to the outer membrane leaflet, assessed by DPA translocation experiments. Consistent with the expected lack of channel block, DPA antagonism did not interact with permeant ions. Therefore, we speculate that voltage dependence may arise from interactions of DPA with the inherent voltage dependence of channel gating. Overall, we conclude that DPA noncompetitively inhibits NMDA-induced current by a novel voltage-dependent mechanism and represents a new class of anionic NMDAR antagonists.