The field of intravenous and intra-arterial thrombolysis for the treatment of acute ischemic stroke is rapidly advancing. Limitations of existing thrombolytic agents have prompted the development of new thrombolytic agents over the last decade. These new agents are broadly classified as third generation thrombolytics. Two of the several third generation thrombolytic agents have been investigated for the treatment of acute ischemic stroke and include tenecteplase (Genentech Inc) and reteplase (Roche Holding AG). By virtue of structural modifications, third generation thrombolytics have longer half-lives and greater penetration into the thrombus matrix. Tenecteplase has been evaluated in experimental models of ischemic stroke. These experimental studies have observed faster and more complete recanalization of occluded arteries compared with second-generation thrombolytics. The first prospective human clinical trial evaluated the safety and efficacy of intra-arterial reteplase in 16 patients with ischemic stroke who were poor candidates for intravenous alteplase therapy. Near complete or complete recanalization was observed after treatment in 88% of the patients. The development and use of third generation thrombolytics is expected to increase the rate of recanalization and clinical recovery in patients with ischemic stroke. Clinical trials are required to determine the appropriate dose and patient selection for these emerging pharmacological agents.