Acetyl-L-carnitine (ALCAR), a guanidino compound, is reported to be neuroprotective against ischemia-reperfusion injury (IRI).1,2 Energetically, ALCAR provides acetyl-CoA to TCA cycle to maintain aerobic energetic metabolism in electron-transport system. In addition, it is speculated that ALCAR inhibits peroxidation of tissue as an antioxidant. The mechanism of neuroprotective effects of ALCAR was investigated by spin resonance analyses; phosphorous NMR (31P-NMR)3,4 and electron spin resonance spectroscopy (ESR). 4 Wistar rats (male, 6-wks, 200g, n=5 per group) were used. In each experiment, a rat was deeply anaesthetised with diethyl ether and then decapitated. 400-μm-thick brain slices were loaded into a NMR sample tube and superfused with well oxygenated ACSF with ALCAR (0, 0.1, 1 mM) at 27.5°C. High-energy phosphates in brain slices, phosphocreatine (PCr) and ATP, were non-destructively measured by 31P-NMR (DRX300wb, Bruker, Germany). Brain slices were exposed to IRI by halting the perfusion for 1 hr, followed by the reperfusion for 3 hrs. The ESR measurement of the radical scavenging activity of ALCAR was based on the competitive reaction with spin trapping agent DMPO or g-CYPMPO.5 Antioxidant activity of ALCAR on peroxidation in brain homogenate was also evaluated by thiobarbituric acid reactive substances (TBARS) assay. All experiments were repeated 3-5 times. Values are means ± SEM, compared by ANOVA. 31P-NMR demonstrated significantly better recovery of PCr in brain slices superfused with 0.1 mM ALCAR (72±5%) than control (59±1%; p<0.05). But no better recovery was observed with 1 mM ALCAR (59±3%). In ESR study, ALCAR exhibited direct radical scavenging activity for ascorbate free radicals (EC50 0.34 mM), DPPH radicals and methyl radicals in dose-dependent manners (p<0.05). On the other hand, ALCAR at mM-order concentrations increased amounts of superoxide anion (O2●-) and hydroxyl radicals (●OH) than control. TBARS assay demonstrated that ALCAR inhibited peroxidation by carbon-centered radicals at concentrations between 0.1 μM and 1 mM (p<0.05), whereas peroxidation was doubled at 10 mM. ALCAR did not inhibit peroxidation by ●OH. In conclusion, it is speculated that ALCAR might be neuroprotective against IRI in a limited range of concentration (i.e. μM-order) through its antioxidant activity attributable to its free radical scavenging activity. Interestingly, ALCAR increased amounts of O2●- and ●OH at concentrations in mM-order which might support the findings by NMR and TBARS assay that ALCAR might not be neuroprotective at mM-order concentration. Supported by JSPS KAKENHI to OT (#24592304), TK (#23592259 and IY (#23592099).