Long-term cultures of human corneal endothelial cells have been established. In culture, these cells form a dense monolayer (about 500,000 cells cm-2), similar to that found in vivo, and synthesize an extracellular matrix containing laminin, entactin, and fibronectin. Factor VIII and angiotensin-converting enzyme were not found in either the cultured or native corneal endothelium. Cells were obtained by scraping corneal buttons that had been preincubated in the culture medium supplemented with endothelial cell mitogen. The human corneal endothelium was grown under conditions virtually the same as those used for cultivation of human vascular endothelial cells, namely, on fibronectin- or gelatin-coated tissue culture plastic in Medium 199 supplemented with 20% human serum and 400 micrograms ml-1 endothelial cell growth supplement. Human corneal endothelial cells from the culture obtained can be used for transplantation onto human corneas, for studying repair of damaged corneal endothelium in situ, as well as for in vitro studies of cell growth regulation.