Extracellular glutamate is kept below a toxic level by glial and neuronal glutamate transporters. Here we show that the transportable glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC) induced cell death in mature, but not in immature, hippocampal neuron-enriched cultures. The cell death produced by a 24-hr treatment with t-PDC was dose-dependent and reached 85% of the cell population at a 250 microM concentration at 23 days in vitro (DIV). Immunocytochemistry experiments showed that, under these experimental conditions, t-PDC killed not only neurons as expected but also glial cells. The N-methyl-D-aspartate (NMDA) antagonist D-2-aminophosphonovalerate (D-APV; 250 microM) only partially reversed this toxicity, completely protecting the neuronal cell population but not the glial population. The antioxidant compounds alpha-tocopherol or Trolox, used at concentrations that reverse the oxidative stress-induced toxicity, did not block the gliotoxicity specifically produced by t-PDC in the presence of D-APV. The nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) elicited cell death only in mature, but not in immature, hippocampal cultures. The TBOA toxic effect was dose dependent and reached a plateau at 100 microM in 23-DIV cultures. About 50% of the cell population died. TBOA affected essentially the neuronal population. D-APV (250 microM) completely reversed this toxicity. It is concluded that nontransportable glutamate uptake inhibitors are neurotoxic via overactivation of NMDA receptors, whereas transportable glutamate uptake inhibitors induce both an NMDA-dependent neurotoxicity and an NMDA- and oxidative stress-independent gliotoxicity, but only in mature hippocampal cultures.