Delayed treatment of stroke with recombinant tissue plasminogen activator (r-tPA) induces overexpression of matrix metalloproteinase 9 (MMP-9) which leads to breakdown of the blood–brain barrier (BBB) and causes more injuries to the brain parenchyma. In this study, the effect of ascorbic acid (AA), an antioxidant agent, on the delayed administration of r-tPA in a rat model of permanent middle cerebral artery occlusion (MCAO) was investigated. Forty male rats were randomly divided into four groups: untreated control rats (ischaemic animals), AA-treated (500 mg/kg; 5 hr after stroke) rats, r-tPA-treated (5 hr after stroke 1 mg/kg) rats and rats treated with the combination of AA and r-tPA. Middle cerebral artery occlusion was induced by occluding the right middle cerebral artery (MCA). Infarct size, BBB, brain oedema and the levels of MMP-9 were measured at the end of study. Neurological deficits were evaluated at 24 and 48 hr after stroke. Compared to the control or r-tPA-treated animals, AA alone (p < 0.001) or in combination with r-tPA (p < 0.05) significantly decreased infarct volume. Ascorbic acid alone or r-tPA + AA significantly reduced BBB permeability (p < 0.05), levels of MMP-9 (p < 0.05 versus control; p < 0.01 versus r-tPA) and brain oedema (p < 0.001) when compared to either the control or the r-tPA-treated animals. Latency to the removal of sticky labels from the forepaw was also significantly decreased after the administration of AA + r-tPA (p < 0.05) at 24 or 48 hr after stroke. Based on our data, acute treatment with AA may be considered as a useful candidate to reduce the side effects of delayed application of r-tPA in stroke therapy.