Defects in neurotransmitter glutamate transport may be an important component of chronic neurotoxicity in diseases such as amyotrophic lateral sclerosis. There are no reliable models of slow glutamate neurotoxicity. Most previous in vitro systems have studied the rapid neurotoxic effects of direct-acting glutamate agonists. Therefore, we developed a model of slow toxicity in cultured organotypic spinal cord slices. The model was based on selective inhibition of glutamate transport, which continuously raised the concentration of glutamate in the culture medium. This resulted in the slow degeneration of motor neurons over several weeks. Motor neuron toxicity was selectively prevented by non-N-methyl-D-aspartate glutamate receptor antagonists and glutamate synthesis or release inhibitors but not by N-methyl-D-aspartate receptor antagonists. Thus, selective inhibition of glutamate transport produces a model of clinically relevant slow neurotoxicity and appears to be mediated by the action of non-N-methyl-D-aspartate receptors. This data supports the hypothesis that the slow loss of motor neurons in amyotrophic lateral sclerosis could be due, in part, to defective glutamate transport.