This study examined the effects of an inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis, N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011), on infarct volume, volume at risk, cerebral blood flow (CBF), and levels of cytochrome P450 (CYP450) eicosanoids in the brain after transient occlusion of the middle cerebral artery (t-MCAO) in rats. TS-011 (0.1 mg/kg, iv) reduced cortical infarct volume by approximately 70% and total infarct volume by 55%. TS-011 had no effect on the volume at risk or CBF during or up to 30 mins after the ischemic period. TS-011 reduced the delayed fall in CBF seen 2 h after reperfusion. The levels of CYP450 eicosanoids were similar in the ischemic and contralateral hemispheres after t-MCAO. TS-011 reduced 20-HETE levels in cerebral tissue by 80% but had no effect on the levels of EETs. Administration of another 20-HETE inhibitor, HET0016 (0.01 to 1.0 mg/kg, iv) or a 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (10 mg/kg, iv) also reduced infarct size. These results indicate that inhibitors of the synthesis or vasoconstrictor effects of 20-HETE reduce infarct size in rats after cerebral ischemia. The effects of TS-011 are not associated with changes in the area at risk or CBF and may be because of a potential protective effect in neurons subjected to ischemic stress.