Endothelial cells involved in tumor angiogenesis, wound healing, and inflammation are predominantly of microvascular origin and are functionally distinct from large vessel-derived endothelial cells which have been largely used for in vitro vascular research. To overcome the problems commonly involved in the culture of microvascular endothelial cells, including unreliable isolation techniques and low cell yields, we developed a simplified protocol for the selective cultivation of human dermal microvascular endothelial cells (HDMEC) obtained from neonatal foreskins, based on the transient, endothelial cell-specific induction of E-selection by tumor necrosis factor-alpha (TNF-alpha). Subconfluent primary cultures, consisting of a mixture of endothelial cells, fibroblasts, and keratinocytes, were treated with TNF-alpha for 6 h, and HDMEC were isolated by their selective binding to magnetic beads coupled with anti-E-selection monoclonal antibody. After two immunomagnetic purification steps, a homogenous population of HDMEC was obtained which showed typical cobblestone morphology, expressed CD31 and von Willebrand factor, proliferated in response to vascular endothelial growth factor, upregulated the expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 in response to TNF-alpha, and formed capillary-like tubes in a three-dimensional collagen type I matrix. This simple technique may facilitate a more widespread use of microvascular endothelial cell cultures obtained from different human or animal organs for functional in vitro studies.