Background and Purpose — Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) after stroke increases risk of hemorrhagic transformation, particularly in areas with blood-brain barrier leakage. Our aim was to characterize acute effects of rtPA administration on the integrity of microvascular barriers. Methods — Stroke was induced in spontaneously hypertensive rats by unilateral embolic middle cerebral artery occlusion. Six hours after stroke, rtPA was intravenously administered (n=10). Controls received saline (n=4). Extravasation of the large-diameter contrast agent monocrystalline iron oxide nanocolloid (MION) was assessed with susceptibility contrast-enhanced MRI during rtPA injection. In addition, we performed perfusion MRI and diffusion-weighted MRI. After MRI, 2 hours after rtPA treatment, intracerebral hemorrhage was quantified with a spectrophotometric hemoglobin assay. Results — Late rtPA treatment resulted in increased hemorrhage volume (8.4±1.7 versus 2.9±0.9 μL in controls; P <0.05). In MION-injected animals, during rtPA administration, transverse relaxation rate change (ΔR ₂ *) increased from 12.4±6.0 to 31.6±19.2 s ⁻¹ ( P <0.05) in areas with subsequent hemorrhage. Significant ΔR ₂ * changes were absent in nonhemorrhagic areas, in animals without injected MION, and in saline-treated animals. Thrombolytic therapy did not improve perfusion in regions with hemorrhagic transformation (cerebral blood flow index was 22.8±19.7% [of contralateral] at 0.5 hours before and 22.4±18.0% at 1 hour after rtPA administration). Conclusions — The ΔR ₂ * changes during rtPA delivery in MION-injected animals indicate extravasation of MION, which reflects increased permeability of the blood-brain barrier. This implies that late rtPA treatment rapidly aggravates early ischemia-induced damage to microvascular barriers, thereby enhancing hemorrhagic transformation.