Tenecteplase (TNK) was engineered to have increased fibrin specificity and an increased half-life compared to Alteplase. Although Tenecteplase is currently being tested in a Phase II clinical trial in acute ischemic stroke patients, little is known about the pharmacology and dose-response or therapeutic window for Tenecteplase in embolic stroke models. In the present study, we compared Tenecteplase with Alteplase on behavioral outcome in rabbits with embolic strokes. Male New Zealand white rabbits were embolized by injecting a suspension of small blood clots into the middle cerebral artery (MCA) via a catheter. The rabbit small clot embolic stroke model (RSCEM) was used for a dose-response profile analysis of Tenecteplase (0.1 mg/kg-3.3 mg/kg) and Alteplase (0.9 mg/kg-3.3 mg/kg) given intravenously 1 h following embolization. In additional studies, Tenecteplase (0.9 mg/kg) or Alteplase (3.3 mg/kg) was administered 3 (or 6) h following embolization to determine the therapeutic window for the thrombolytics. For both studies, behavioral analysis was conducted 24 h following embolization, allowing for the determination of the effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a drug is considered beneficial if it significantly increases the P50 compared with the control group. The P50 of controls 24 h after embolization was 1.13 +/- 0.15 mg. Rabbits treated 1 h post-embolization with Tenecteplase (0.1, 0.25, 0.9, 1.5 or 3.3 mg/kg) had P50 values of 1.48 +/- 0.33, 2.20 +/- 0.44, 2.76 +/- 0.37, 2.15 +/- 0.29 and 2.78 +/- 0.31 mg, respectively. In Alteplase-treated rabbits, only the 3.3 mg/kg dose significantly increased the group P50 by 189% compared to control. Tenecteplase was also effective at increasing the P50 value to 2.21 +/- 0.43 mg if there was a 3-h delay following embolization, but not if there was a 6-h delay before administration. Alteplase was only effective if administered 1 h following embolization where it significantly increased the P50 value to 3.27 +/- 0.40 mg. This study indicates that Tenecteplase has a wide therapeutic range, a therapeutic window of at least 3 h and a durable effect. Moreover, the safety profile for Tenecteplase is similar to that of Alteplase. Tenecteplase does not increase the rate of intracerebral hemorrhage (ICH) above that produced by Alteplase. However, the therapeutic range and window for Alteplase is more limited than that for Tenecteplase. Our preclinical studies suggest that Tenecteplase has a better pharmacological profile than Alteplase and supports further investigation of Tenecteplase in randomized double-blinded clinical trials in stroke patients.