Published methods for the isolation of cerebral microvessels primarily yield terminal resistance vessels and capillary networks, not the more proximal, subpial penetrating arterioles desired for certain studies. We report a novel method for isolating microvessels from the cerebral cortex of a single guinea-pig brain that yields large arteriolar complexes that are up to 50% intact. Instead of using homogenization to disperse brain parenchyma, we digested cortical fragments with trypsin, gently dispersed the parenchyma mechanically, and recovered microvascular complexes by sieving. Phase-contrast and electron microscopy showed primary (penetrating) arterioles, secondary arterioles, and capillary networks that frequently were in continuity as intact microvascular units. Culture of microvascular cells was carried out by enzymatic dissociation followed by an overnight incubation in a recovery medium at 4°C before plating onto fibronectin-modified surfaces. Viability of isolated cells was demonstrated by good cell attachment and prompt proliferation that resulted in confluent cultures after 10 days. Confluent secondary cultures demonstrated characteristic features of smooth muscle cells, including a “hill-and-valley” growth pattern and expression of α-actin. Less than 1% of cells were endothelial or astrocytic cells by immunocytochemical and morphologic criteria. Ultrastructural studies demonstrated evidence of a synthetic phenotype of smooth muscle cell and absence of a significant number of fibroblasts. This method demonstrates that viable smooth muscle cells from the cerebral parenchymal microvasculature can be isolated in bulk quantities for study in vitro.