Involvement of adenosine and adenosine triphosphate-sensitive potassium (KATP) channels in the development of ischemic tolerance has been suggested in global ischemia, but has not been studied extensively in focal cerebral ischemia. This study evaluated modulating effects of adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and mitochondrial KATP channel blocker 5HD (5-hydroxydecanoate) on the development of tolerance to focal cerebral ischemia in rats. Preconditioning with 30-minute middle cerebral artery occlusion (MCAO) reduced cortical and subcortical infarct volume following 120-minute MCAO (test ischemia) given 72 hours later. The neuroprotective effect of preconditioning was attenuated by 0.1 mg/kg DPCPX given before conditioning ischemia (30-minute MCAO), but no influence was provoked when it was administered before test ischemia. DPCPX had no effect on infarct volume after conditioning or test ischemia when given alone. The preconditioning-induced neuroprotection disappeared when 30 mg/kg 5HD was administered before test ischemia. These results suggest a possible involvement of adenosine A1 receptors during conditioning ischemia and of mitochondrial KATP channels during subsequent severe ischemia in the development of tolerance to focal cerebral ischemia.