Oxidative stress (OS) is implicated in Amyotrophic Lateral Sclerosis (ALS), a progressive and fatal neurodegenerative disease (Cookson and Shaw, 1999). The present study was designed to assess the functional changes in motor neurones (MNs) following exposure to OS using cell-attached patches to measure the effects of H₂O₂ on both cellular excitability and single channel activity. Spinal cord cultures were grown from humanely killed E13 mice (Boolaky et al. 2002), MNs being identified morphologically and immunohistochemically. Whole-cell and cell-attached recordings were made. The bath solution contained (mM): 140 NaCl; 3 KCl; 2 MgCl₂; 2 CaCl₂; 10 HEPES; 10 Glucose, pH 7.4. The pipette solution contained (mM): 140 KCl; 5 NaCl; 1 MgCl₂; 1 CaCl₂; EGTA 11; HEPES 10, pH 7.2. In single-channel experiments 1 μM TTX was added to the bath solution. Cells were patched under control conditions and following incubation for 1 hour (5% CO₂, 37^oC) in medium containing 100μM H₂O₂. All recordings were made at 0mV pipette potential (pp). Electron paramagnetic resonance (EPR) spectroscopic detection of α-phenyl-tert-butylnitrone adducts was incorporated for direct detection of free radicals. Control cells possessed healthy RMP, and 88 % of cells had spontaneous biphasic action currents. Following treatment with 100μM H₂O₂, both the RMP (Table 1) and the number of cells firing decreased to 59%; the interspike interval, a measure of firing frequency, increased. The Popen of large conductance maxi-K channels, at 0mV pp, was significantly increased in MN (Table 1) and the voltage dependence was decreased following H₂O₂-treatment (n = 4). Incubation with 100μM riluzole did not prevent membrane depolarisation or the reduction in the firing frequency, although it did prevent reduction in the number of cells firing. EPR spectroscopy showed that incubation with 100μM H₂O₂ increased the concentration of lipid-derived free radicals identified as a combination of alkoxyl, alkyl and hydroxyl radical species. However, this was not affected following addition of 100μM riluzole. This study demonstrates that OS induced by H₂O₂ produces significant changes in the excitability of MN in culture. Such changes may mirror the excitability increases in ALS and underpin neurodegeneration