The neuronal death after ischemia is closely linked to the essential role of mitochondrial metabolism. Inhibition of mitochondrial respiratory chain reduces ATP generation leading to a dysregulation of ion metabolism. Acetyl-L-carnitine (ALC) influences the maintenance of key mitochondrial proteins for maximum energy production and it may play a neuroprotective role in some pathological conditions. In this study we have analyzed ALC-mediated neuroprotection on an in vitro model of brain ischemia. Field potential recordings were obtained from a rat corticostriatal slice preparation. In vitro ischemia (oxygen and glucose deprivation) was delivered by switching to a solution in which glucose was omitted and oxygen was replaced with N2. Ten minutes of in vitro ischemia caused an irreversible loss of the field potential amplitude. Pretreatment with ALC produced a progressive and dose-dependent recovery of the field potential amplitude following in vitro ischemia. The neuroprotective effect of ALC was stereospecific since the pretreatment with two different carnitine-related compounds did not cause neuroprotection. The choline transporter inhibitor hemicholinium-3 blocked the neuroprotective effect of ALC. ALC-mediated neuroprotection was also prevented either by the non-selective muscarinic antagonist scopolamine, or by the putative M2-like receptor antagonist methoctramine. Conversely, the effect of ALC was not altered by the M1-like receptor antagonist pirenzepine. These findings show that ALC exert a neuroprotective action against in vitro ischemia. This neuroprotective effect requires the activity of choline uptake system and the activation of M2 muscarinic receptors.