Microvascular endothelial cells from rat lungs were cultured in serum-free medium supplemented with an endothelial growth substance, insulin, hydrocortisone and so on. Five to seven days after plating, cultured cells formed a monolayer. They were identified as endothelial cells by morphology and by positive immunohistochemistry for factor VIII-related antigen, a marker for endothelia cells. Differences between gelatin coated culture plates and plastic culture plates in endothelial cell proliferation were evaluated. Cells plated on uncoated plastic plates had a spindle-shaped morphology and did not express factor VIII-related antigen. Two types of medium, serum-free medium containing endothelial growth substance and basal medium supplemented with 20% fetal calf serum, were also compared in primary culture. In contrast with the serum-free medium, cells cultured in the serum-containing medium showed fibroblast-like morphology and did not express factor VIII-related antigen. These results suggest that a gelatin substratum and serum-free medium containing endothelial growth supplement are necessary for in vitro proliferation of microvascular endothelial cells isolated from rat lungs. The culture method and conditions outlined here allow the proliferation of pure microvascular endothelial cells from rat lungs. It may be useful in studying hematogenous metastasis to the lung and the role of microvascular endothelium in other pulmonary disease.