A new method to study the interaction of astrocytes and pericytes with cerebral capillary endothelial cells in vitro is described. Endothelial cells derived from bovine brain were cultured on gelatin coated slides and covered with type 1 collagen. Endothelial cells aggregated and formed capillary-like structures (CS) within 3 days. The lining cells of the CS stained immunohistochemically for factor VIII-related antigen. Astrocytes isolated from neonatal mice or pericytes from bovine brain were added to the preparations after the formation of CS. After various periods of co-culture, the slides were fixed with methanol and examined with the immunohistochemical stain for glial fibrillary acidic protein or smooth muscle actin to demonstrate astrocytes or pericytes respectively. Five hours after addition, only 10% of astrocytes were associated with CS. However, by 24 hours, 70% of the astrocytes had assumed a position adjacent to the CS. The astrocytes then developed processes which were intimately apposed to the CS by 3 days, at which time they resembled the in vivo structural relationship between astrocytes and microvessels that occur in areas of central nervous system injury. Progressive elongation of the astrocytes or their processes at the CS was evident at 6 and 9 days of co-culture. The cross-section of CS co-cultured with astrocytes showed continuous cells surrounding a lumen, and the endothelial cells appeared to be connected by tight junctions. When pericytes were added to CS cultures they also preferentially associated with CS, but the contact occurred more rapidly than with astrocytes, 50% being associated with CS by 5 hours. The CS were almost completely covered with elongated pericytes by 24 hours. A chemotactic assay was developed that showed that there was a chemotactic attraction of pericytes to the CS. Thus an in vitro system is now available to study the interrelationships of these cell types and their interaction in development, regeneration and differentiation of the blood-brain barrier.