Porcine cerebral microvascular (PCMV) endothelial cell cultures and pericyte-endothelial cell cocultures were established and the self-organizational properties of the cells were examined in various culture conditions. Cultured PCMV endothelial cells were characterized by the capacity to produce prostacyclin in response to bradykinin. Cultured PCMV pericytes were identified with a smooth muscle actin-specific stain. PCMV endothelial cells organized into cord structures when left in culture for several weeks without passage. Lumina were observed in cross sections of these cords and appeared to form through a process of cell-selective autolysis. PCMV endothelial cells required three dimensions for self-organization, forming suspended cords in planes that either intersected or paralleled the culture vessel floor. After formation, suspended cords continued to exhibit a morphologic plasticity punctuated by the coordinated migrations of PCMV endothelial cells en masse. Sequential propagation of PCMV endothelial cell monolayers and development of suspended capillarylike cords recurred cyclically when cells were left in culture without passage for several weeks. Cord development was also observed in PCMV pericyte-endothelial cell cocultures with large proportions of pericytes. However, pericytes were not located in cross sections of suspended cords formed in coculture. Apparently, in some conditions of PCMV coculture, populations of PCMV endothelial cells and pericytes segregate. Retina-derived growth factor (RDGF) promoted this cell-type segregation and the subsequent formation of suspended cords in PCMV cocultures, although its exact mode of action is unclear. These results indicate that cultured cerebral microvascular endothelial cells and pericytes have capacities for complex, temporal self-organization that varies according to culture conditions.