Low doses of thrombin are neuroprotective while high doses are neurotoxic and lead to brain injury. However, evidence suggests that low doses of thrombin cause brain injury when infused concomitantly with tissue plasminogen activator (tPA), which is used clinically to facilitate evacuation of intracerebral hematomas. In this study, we examined the effects of intracerebral infusion of tPA and thrombin, individually and in combination. Rats were infused in the right basal ganglia with 50 microL saline solutions containing thrombin, tPA, or thrombin + tPA. In the first experiment, rats were used for blood-brain barrier (BBB) permeability measurements at 24 h after infusion. In the second experiment, animals were euthanized 3 days after infusion, and brain sections were stained with Fluoro-Jade to measure neuronal cell death. Behavioral tests were carried out before and after surgery. Infusion of thrombin + tPA markedly increased Evans blue tissue content in ipsilateral brain samples (p < 0.05). Fluoro-Jade-stained sections from thrombin + tPA group demonstrated significantly higher cell death counts (p < 0.01). Significant neurological deficit was revealed in thrombin + tPA group in forelimb-placing and corner-turn tests (p < 0.01). This study shows that tPA potentiates the neurotoxic effects of thrombin and leads to increased BBB permeability, neuronal cell death, and neurological deficit. Our results suggest that using tPA to lyse intracerebral hematomas has potential to produce neuronal cell death and disruption of BBB.