The treatment of acute ischemic stroke (AIS) using thrombolysis with recombinant tissue-plasminogen activator (rtPA, alteplase) is limited by its narrow time window and the risk of hemorrhage. Recombinant plasminogen activator (rPA, reteplase) has been used clinically on coronary artery thrombosis and acute myocardial infarction. It is necessary to induce strokes experimentally as a means of validating the rPA timing on patients with AIS. However, current embolic models cannot mimic clinical situations well due to the embolus's composition of dried blood clots or artificial materials. In this paper, we used two novel rat thromboembolic models to determine the dosage-effect relationship and therapeutic time window of r-PA. Male rats were administered rPA or rtPA intravenously at 2–12 h postischemia. Cerebral blood flow, behavioral outcomes and infarct volume within the same animal group were determined. Our results demonstrated that rPA (0.2 and 0.4 mg/kg) or rtPA (0.2 mg/kg) restored focal perfusion, reduced cerebral infarction, and improved behavioral outcomes at 2–4 h postischemia. rPA but not rtPA significantly restored focal perfusion at 6 h postischemia. However, delayed rPA-treatment neither decreased infarct volume nor improved the neurological disorder. Cerebral hemorrhage occurred at 6 h postischemia detected by Evan's blue leakage and tissue hemoglobin content. Collectively, Thrombolysis with rPA may be beneficial in revascularization at an acceptable dosage of 0.2–0.4 mg/kg within 6 h after the cerebral infarct onset.