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Papaverine is a non-specific vasodilatory drug, which was found to unexpectedly cause opening of the blood–brain barrier (BBB). We studied the expression of two endothelial cell related markers, endothelial barrier antigen (EBA) as a marker of BBB intactness, and matrix metalloproteinase-9 (MMP-9), an extracellular matrix molecule, the activation of which results in BBB opening. Immunoreactivity was quantified after intra-carotid injection of papaverine in rats. BBB opening was identified at 1 h, but had been reversed by 24 h. Reduction in EBA-immunoreactivity occurred at 3 h, whereas MMP-9 was not observed until 24 h. The activation of MMP-9 thus occurred much later than BBB opening, which suggests a different role for MMP-9 when selective and reversible opening of the BBB is produced by pharmacological manipulation.