This work describes the formation, perfusion, and maturation of three-dimensional microvascular tubes in vitro. These tubes consisted of confluent monolayers of human endothelial cells that lined open, cylindrical channels within collagen gels. Perivascular cells could be directly embedded within the gels or added after endothelial cells grew to confluence. The tubes spanned the entire 5-7 mm extent of the gels; their diameters initially ranged from 55 to 120 microm and increased to 75-150 microm after maturation. Endothelial tubes displayed a strong barrier function over 5 days, resisted adhesion of leukocytes, and reacted quickly to inflammatory stimuli by breakdown of the barrier and support of leukocyte adhesion. These tubes resembled venules and "giant" capillaries in both their cellular organization and function, and we believe that they will serve as useful in vitro models of inflammation under constant perfusion.