We have investigated the interactions of six analogues of pentamidine with the N-methyl-D-aspartate (NMDA) receptor complex. All six compounds were effective NMDA receptor antagonists based upon their ability to inhibit [3H]dizocilpine binding to rat brain membranes. IC50 values ranged from 2 to 18 microM, and all compounds had Hill coefficients in excess of 1 suggesting a non-competitive interaction with [3H]dizocilpine. All compounds also inhibited NMDA- and glycine-induced intracellular Ca2+ changes measured in cultured rat forebrain neurons using the fluorescent indicator, fura-2. IC50 values in this assay ranged from 0.4 to 4.7 microM. Whereas pentamidine is directly toxic to cultured neurons, this was not a consistent finding with the pentamidine analogues tested, indicating that the toxic effects are not related to NMDA receptor antagonism. Finally, all of the agents tested were also effective in protecting neurons from NMDA-induced neurotoxicity. These data emphasize the possible utility of pentamidine-like drugs as neuroprotective agents and suggest that it is possible to generate compounds with a wider margin of safety than pentamidine itself.