We describe a new technique for isolating microvessels from both brain and brain tumors. This method is relatively quick and provides a microvessel preparation free of contamination by other brain tissue. Using this method, structurally intact microvessels from normal rat brain and from a malignant rat astrocytoma were isolated and characterized with light microscopy, scanning electron microscopy and transmission electron microscopy. In contrast to microvessels derived from normal rat brain, rat astrocytoma microvessels had endothelial cells with multilayered basement membranes, extensive microvilli on the cell surfaces, and a significant increase in the number of pinocytes in the cytoplasm. Furthermore, astrocytoma microvessel endothelial cells had pleomorphic electron dense nuclei with pale perichromatin, whereas the nuclei of endothelial cells of microvessels derived from normal brain tissue were finely granular and homogeneous with characteristically electron dense perichromatin. The morphologic characteristics of the astrocytoma microvessels are similar to the histologic changes seen in astrocytoma tissue in situ, and correlate well with the known altered functions of brain tumor neovasculature.