Abstract: A dramatic loss of glutamate transport has been observed in sporadic amyotrophic lateral sclerosis and has been postulated to contribute to the disease. Experimentally, this hypothesis was corroborated by mimicking the chronic loss of glutamate transport in post-natal rat spinal cord organotypic cultures through the use of glutamate transport inhibitors. This system is characterized by a relatively selective slow loss of ventral horn motor neurons resulting from glutamate transport inhibition. In this study, spinal cord organotypic cultures were used to test various drugs to evaluate their neuroprotective properties against this slow glutamate-mediated neurotoxicity. The most potent neuroprotectants were drugs that altered glutamate neurotransmission, including non-NMDA receptor antagonists (GYKI-52466, PD144216, and PD139977) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N-t-butyl-α-phenylnitrone) and inhibitors of nitric oxide synthesis (NG-monomethyl-l-arginine acetate) were modestly neuroprotective. The calcium endonuclease inhibitor aurintricarboxylic acid and the calcium release inhibitor dantrolene also provided partial motor neuron protection. However, several antioxidants and calcium channel antagonists had no excitotoxic neuroprotectant activity. This system provides a preclinical screening method for the burgeoning number of drugs postulated for clinical trials in motor neuron disease and a model to evaluate the mechanisms of chronic glutamate toxicity.