Oxygen-derived free radicals are implicated in hypoxia- and reoxygenation-related brain injury. In addition, excitatory amino acid neurotransmitters seem to be involved in this neurotoxicity and could act through the L-arginine/nitric oxide (NO) synthase pathway. In the present study we have used rat forebrain neurons in culture submitted to hypoxia/reoxygenation to investigate the relative role of free radicals, glutamate, and nitric oxide in hypoxic neuronal injury. Hypoxia (5 h) followed by reoxygenation (0-24 h) induced cell damage assessed by lacticodehydrogenase release into culture medium. Superoxide dismutase (SOD, 500 U/mL), D-L-2-amino-5-phosphonovaleric acid (100 microM), a glutamate receptor antagonist, and NG-nitro-L-arginine (100 microM), an NO synthase inhibitor, protected the neurons. The effect of NG-nitro-L-arginine was reversed by adding L-arginine (10 mM) in the culture medium, and hemoglobin, which scavenges NO, also afforded protection. Hypoxia (5 h) provoked glutamate release from neurons, and this effect was inhibited by SOD. Exogenous glutamate (1-100 microM) induced lacticodehydrogenase release, and this effect was inhibited by glutamate antagonism, NO synthase inhibition, or superoxide radical scavenging. These data are consistent with the following sequence of events in hypoxia-related neurotoxicity: free radical formation, glutamate release, and activation of NO synthase leading to superoxide and NO cooperative toxicity.