Human endothelial cells, when suspended within three-dimensional collagen matrices, develop intracellular vacuoles that coalesce to form capillary lumens and tubes. Vacuole and lumen formation are completely dependent on the collagen-binding integrin α2β1, while other endothelial cell integrins had no apparent influence. Vacuole formation occurs by a pinocytic process with internalization of plasma membrane and molecules from the extracellular space, such as fluorescent tracers. By immunofluorescence, vacuole membranes were found to contain associated cell surface proteins, proteins involved in endosomal trafficking (i.e., caveolin and annexin II), and F-actin. Furthermore, some vacuole compartments contained von Willebrand factor. Integrin-regulated vacuole formation and coalescence are major mechanisms controlling capillary lumen and tube formation within a three-dimensional extracellular matrix.