Abstract ONO-2506, a novel agent which modulates miscellaneous functions of cultured astrocytes was shown to abolish delayed infarct expansion, while improving the neurological deficits following permanent focal ischemia in rats. The present study aimed to clarify its therapeutic time window and the mechanism underlying its beneficial effects. Treatment started at 24 and 48 h after the onset of ischemia significantly decreased the infarct volume at 168 h, whereas that started at 72 h was ineffective. In the former experimental group, the neurological deficits were significantly improved since 72 h on. Next, using a transient focal ischemia model in rats, the effect of the agent on the extracellular glutamate level in the periinfarct area was examined by microdialysis. During the period between 22 and 26 h after ischemia, the glutamate level as measured by the OPA-HPLC method markedly increased in the saline-treated group, whereas it remained close to normal in the treatment group. The glial glutamate transporter inhibitor-induced increases in the glutamate level were much greater in the drug-treated group than in the saline-treated group. Our results show that ONO-2506 has a wide therapeutic time window and is capable to induce rapid symptomatic improvement through preservation of the activities of glial glutamate transporters.