We have previously characterized morphologic features of wounding-induced angiogenesis that occurs in response to acute and chronic gastric mucosal injury. As a means of investigating the molecular mechanisms underlying gastric angiogenesis, microvascular endothelial cells were isolated from stomachs of normal (non-injured) rats. The isolation procedure adapted and combined aspects of previous methods and employed positive selection using magnetic beads coated with monoclonal antibody specific for rat CD31 (PECAM-1), a cell surface marker restricted to platelets, monocytes, T lymphocytes and endothelial cells. The isolated microvascular endothelial cells expressed vascular endothelium-specific antigen and the endothelial-specific receptors, Tie2 and flt-1 (VEGFR1). When plated on growth factor-reduced matrigel, the isolated microvascular endothelial cells formed capillary-like structures reflecting in vitro angiogenesis. These cells were also responsive to vascular endothelial growth factor, VEGF, further verifying their endothelial nature. The rat microvascular endothelial cells isolated by this procedure should be useful in delineating molecular mechanisms and regulation of the angiogenesis that is essential for the healing of acute and chronic gastric injury.