Previous work has shown that tissue plasminogen activator (tPA) is a key enzyme in the control of fibrinolysis within the vascular system. The sources of brain tPA and the mechanisms by which tPA secretion and production occur within cerebral microcirculation are not well established. In this study, expression of tPA was investigated in cerebral capillaries and capillary-depleted brain isolated from cortices of 4- to 5-week-old rats and guinea pigs. In both species, a single tPA band of M(r) 67,000 was detected in cerebral capillaries by Western blot analysis. The tPA signal was absent from capillary-depleted brain. These results were corroborated at the messenger ribonucleic acid level. Reverse transcription-polymerase chain reaction analysis revealed the presence of tPA complementary deoxyribonucleic acid in samples derived from cerebral microvessels and demonstrated very low or undetectable tPA expression in capillary-depleted brain. Immunohistochemical analysis confirmed tPA localization in endothelial cells of brain capillaries. We conclude that microvascular endothelium, i.e., the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation. Delineation of the molecular mechanisms of blood-brain barrier-mediated fibrinolysis will likely contribute to future stroke prevention efforts.