The neuronal toxicity of potassium cyanide (KCN) and 3-nitropropionic acid (3-NPA) has been shown to involve the NMDA receptor (1, 2), but it is not clear whether the same downstream effectors of NMDA-induced neurotoxicity are also involved in the toxicity of both compounds. Excessive stimulation of the NMDA receptor elevates intracellular calcium levels, resulting in the generation of reactive oxygen or nitrogen species such as superoxide anion, hydrogen peroxide, and nitric oxide, which are known to mediate neuronal death (3). We hypothesized that similar downstream effectors of NMDA receptor-mediated neuronal damage may be involved in the induction of neurotoxicity by both poisons. Cerebellar granule neuronal cultures from 7- or 8-day old Sprague-Dawley rat pups were prepared as earlier described (4), and maintained in Minimum Essential Medium supplemented with 10% Foetal Calf Serum, 2mM glutamine, 25mM KCl, and 50µg/ml gentamicin, at 37oC in a humidified atmosphere of 5% CO2/95% O2. Cells were seeded at 1 x 106 cells/ml into 96-well plates pre-coated with poly-D-lysine. Cytosine arabinoside (10µM) was added to the cultures 24 h after plating to inhibit non-neuronal growth. Treatments were applied at 8 or 9 days in vitro and cultures allowed to recover for at least 18 h before viability was determined using the Alamar Blue reduction assay (5) (incubation period of 6 h). Exposure to the various agents was for 1 or 6 h, and in the case of NMDA receptor antagonists, a pre-treatment period of 15 min was included. Morphological effects were monitored using the Olympus DP50 inverted phase-contrast microscope. Potassium cyanide (10µM – 1mM) lowered neuronal viability in a concentration-dependent manner, and 3-NPA (10µM – 1mM) in a concentration- and time-dependent manner, compared to the controls. The non-competitive and competitive NMDA receptor antagonists, MK-801 (10µM) and D-AP5 (50µM), respectively, blocked completely the reductions in viability induced by both poisons, whereas the glycine-site blocker kynurenic acid (up to 1mM) had no effect. The non-NMDA receptor blocker CNQX (10µM) also failed to modify the toxicity elicited by the poisons. The nitric oxide synthase inhibitor L-NAME (1mM), and the antioxidant enzymes catalase and superoxide dismutase (each at 100U/ml) partially but significantly protected against the neurotoxicity. Morphological effects correlated with results obtained from viability assays. Taken together, these results show that the mechanisms of toxicity of potassium cyanide and 3-nitropropionic acid involve similar pathways downstream of NMDA receptor activation, and also suggest that activation of the glycine-sensitive site on the receptor may not be obligatory for the induction of neurotoxicity by both mitochondrial poisons.