Electron microscopic studies have offered presumptive evidence that the origin of both cellular and extracellular components of the retrocorneal membrane (RCM) derive from corneal endothelial cells. In an attempt to demonstrate conclusively the origin of the cell in the RCM, we performed 8-mm exchange, penetrating keratoplasties between male and female rabbits. After allowing for complete healing and using only clear corneas, we produced central RCM by freezing the central cornea after inducing intraocular inflammation. Care was taken to ensure that the membrane was surrounded by normal donor endothelium and Descemet's membrane was intact. Sex chromatin counts of the cells in the RCM of a female host with a male graft showed an average of 2% Barr bodies. Cells in the membrane of a male host with a female graft showed an average of 40% Barr bodies. The experimental and control sex chromatin counts were almost identical. This study provides conclusive evidence that central retrocorneal, fibroblast-like cells and membranes in rabbits are derived from corneal endothelial cells.