Isolated organ preparations represent valuable models for biomedical research, provided that the functional and morphological integrity of vascular and parenchymal compartments is preserved. In this investigation, we have studied the molecular organization of the cerebral microvessels in the isolated guinea-pig brain maintained in vitro by arterial perfusion, a preparation previously proposed as a model of blood-brain barrier (BBB). Using lectin cytochemistry and immunohistochemistry, we examined the microvasculature of the cerebral cortex after 5 h in vitro to assess: (a) the structure of the endothelial glycocalyx at microscopical and ultrastructural level; (b) the distribution of the junctional molecules occludin, ZO-1, PECAM-1 and vinculin; (c) the distribution of basal lamina molecules, such as collagen type IV, laminin and heparan sulfate proteoglycan. All these components of microvessel wall have been previously shown to be vulnerable to ischemic conditions and their organization could be altered in consequence of the transient hypoxia associated with the brain isolation procedure. Our observations demonstrate that the distribution pattern of the molecules considered (i) is comparable to that shown in the cerebral microvasculature of other mammals and (ii) is similar in brains maintained in vitro and in control brains perfused in situ with fixative. The complex of our observation indicates that the molecular organization of the cerebral microvessels is preserved in isolated guinea-pig brain, thus indicating that these preparations can be used to study the cerebrovascular structure and blood-brain barrier function in a variety of experimental conditions.