In cultured spinal cord neurons, we found that blockers of chloride transport (furosemide, a widely used loop diuretic, and the related compounds piretanide and bumetanide, as well as niflumic and flufenamic acids, used as antiinflamatory agents) prevented N-methyl-D-aspartate (NMDA) receptor activation in a dose-dependent manner and are specific for this class of glutamate receptor. Antagonism of NMDA-mediated currents by chloride transport blockers was voltage independent and showed fast on-ff kinetics. The action was noncompetitive with NMDA and did not arise from interaction with the Zn2+ inhibitory site, because blockade of NMDA-induced responses by furosemide and Zn2+ was additive. The inhibition was greater in a low concentration of glycine, but it could not be overcome by increasing the glycine concentration (up to 100 microM). In contrast, the inhibition was attenuated by the polyamine spermine. Because the presence of spermine was not required for inhibition to develop, we conclude that chloride transport blockers are noncompetitive antagonists of the NMDA receptor, likely acting as inverse agonists of the polyamine site. This action may explain the protective effect that has been shown for some of these drugs in neuronal degeneration; because they also prevent neuronal swelling, they may be good starting compounds for synthesis of appropriate therapeutic agents to ameliorate excitotoxicity.