In situ vascular endothelium is characterized by many cytoplasmic vesicles (caveolae) and vacuoles. In venules these are organized into prominent clusters called vesiculo-vacuolar organelles or VVOs. VVOs provide an important pathway for plasma protein extravasation in response to vasoactive mediators. In contrast, cultured endothelial cells isolated from many sources lack VVOs and generally have few caveolae. Our goal was to preserve VVOs in cultured endothelium. Bovine adrenal microvascular endothelial cells (BCEs) cultured on floating Matrigel-collagen Type I gels with vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exhibited typical VVOs by electron microscopy. Both in vivo and in culture VVOs were caveolin-positive by immunoelectron microscopy. On the basis of caveolin immunostaining, VVOs could also be detected by light (confocal) microscopy. When BCEs were cultured without VPF/VEGF, caveolin staining was finely punctate and electron microscopy confirmed the near absence of VVOs. BCE VVOs were sensitive to N-ethylmaleimide. Other types of endothelium cultured on Matrigel-collagen gels with or without VPF/VEGF exhibited few caveolae and no VVOs. Therefore, preservation of VVOs in cultured endothelium required a specific combination of endothelial cells (BCEs), surface matrix (Matrigel-collagen), and growth factor (VPF/VEGF). These endothelial cells should be useful for in vitro studies of trans-endothelial transport.