We present de novo studies and review published efforts from our laboratory, spanning 12 years (from 1988 to 2000), where we have used ultrastructural approaches to study the functional anatomy of the microvasculature in man and animals in health and disease. These efforts have defined a new endothelial cell organelle, termed the vesiculo-vacuolar organelle (VVO), which participates in the regulated transendothelial cell passage of soluble macromolecules. The studies defining this organelle utilized ultrathin serial sections, three-dimensional computer-assisted reconstructions, and ultrastructural electron-dense tracers to establish luminal to abluminal transendothelial cell continuity of VVOs. Commonality of VVOs and caveolae is suggested by the ultrastructural anatomy of individual units of VVOs and caveolae, the presence of caveolin in both structures, and a mathematical analysis of morphometric data, all of which suggest that VVOs form from fusions of individual size units equivalent to vesicles of caveolar size. Ultrastructural studies have localized potent permeability factors and their specific receptors to VVOs in in vivo tumor and allergic inflammation models. Regulation of permeability through VVOs has been quantified and shown to be increased in tumor microvessels and in control vessels exposed to potent permeability-inducing mediators. The transendothelial cell passage of particulate macromolecules occurs by vacuolar transport in tumor vessels; in permeability factor-exposed control vessels, colloidal carbon traversed endothelial cells via the development of pores that did not communicate with or disrupt intercellular junctions by gap formation. Serial section and computer-assisted reconstructions established these findings and suggested the possible development of transendothelial cell pores from VVOs. Serial sectioning and computer-assisted three-dimensional reconstructions of ultrastructural samples of an acute inflammation model revealed a transendothelial cell traffic route for motile neutrophils and platelets in the absence of classical ultrastructural criteria for regulated secretion from either cell.