Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease and the mechanisms leading to selective degeneration of motor neurons are still unknown. Several pathogenetic factors have been proposed, including microglia activation and neuronal hyperexcitability, both observed in mouse models and patients. To study whether the glia cells contribute to the neuronal hyperexcitability observed in cultured cortical neurons from the transgenic mouse model (G93A) of ALS, we performed electrophysiological experiments utilizing the patch clamp technique in coculture of neurons and glial cells. Monolayer cultures of glia from Control and G93A cortices were first prepared and maintained for 21 days and neuron-enriched cell suspensions taken from Control or G93A animals were then added to the Control or G93A glia cultures. After 8–12 days in culture, the neuronal activity was studied by performing current clamp experiments in: glia_Ctrl/neuron_Ctrl, glia_Ctrl/neuron_G93A, glia_G93A/neuron_Ctrl, glia_G93A/neuron_G93A conditions. Interestingly, the number of action potential was not significantly different between glia_G93A/neuron_G93A and glia_Ctrl/neuron_G93A but resulted significantly higher when compared to glia_Ctrl/neuron_Ctrl and glia_G93A/neuron_Ctrl. These data strongly seem to indicate that the cortical hyperexcitability previously observed in G93A cultures was not induced by glia cells but seems to be due to an intrinsic neuronal feature, opening new area of research in the ALS physiopathology.