Purpose: To describe a surgical technique using an artificial anterior chamber to facilitate harvest of Descemet’ s membrane (DM) and endothelium for corneal endothelial cell transplantation. Design: Laboratory investigation. Methods: Corneoscleral buttons of seven human donor eyesweremounted endothelial side up on an artificial anterior chamber. Keeping the endothelial side with its usual concavity, a manual trephination was made on the posterior surface with a 9.0- mm trephine, inside the Schwalbe line and just past the DM in depth. The chamber was filled with air, causing the endothelial side of the donor cornea to assume a convex configuration. The DM along with its endothelium was separated from the posterior stroma using a blunt cyclodialysis spatula. Drops of trypan blue 0.3% and alizarin red S 0.2% (n=6) were applied. The stained DMs were examined under a light microscope and photographed to calculate the percentage of endothelial cell damage. Histology was done on the unstained cornea. Results: The DM carrying endothelium was successfully removed from the posterior stroma in all seven eyes. Although the DM appears to be very friable, all samples were removed in toto without rupture. Vital staining showed amean endothelial cell loss of 8.46% (standard deviation (SD) 6.9). Direct light microscopy demonstrated the preservation of endothelial cell morphology. Conclusions: This technique appears to be a safe and straight for wardmethod to harvest DM for endothelial cell transplantation. Further studies are underway to determine the optimal method of insertion of the obtained healthy DM with endothelial cells through small corneal incisions. Methods: To describe a surgical technique using an artificial anterior chamber to facilitate harvest of Descemet ’s membrane (DM) and endothelium for corneal endothelial cell transplantation. Design: Laboratory investigation. Methods: Corneoscleral buttons of seven human donor eyeswere mounted endothelial side up on an artificial anterior chamber. Keeping the endothelial side with its usual concavity, a manual trephination was made on the posterior surface with a 9.0- mm trephine, inside the Schwalbe line and just past the DM in depth. The chamber was filled with air, causing the The side of the donor cornea to assume a convex configuration. The DM along with its endothelium was separated from the posterior stroma using a blunt cyclodialysis spatula. Drops of trypan blue 0.3% and alizarin red S 0.2% (n = 6) were applied. The stained DMs were examined under a light microscope and photographed to calculate the percentage of endothelial cell damage. Histology was done on the unstaine Results: The DM carrying endothelium was successfully removed from the posterior stroma in all seven eyes. Although the DM appears to be very friable, all samples were removed in toto without rupture. Vital staining showed amean endothelial cell loss of 8.46% ( standard deviation (SD) 6.9). Direct light microscopy demonstrated the preservation of endothelial cell morphology. Conclusions: This technique appears to be a safe and straight for wardmethod to harvest DM for endothelial cell transplantation. Further studies are underway to determine the optimal method of insertion of the acquired healthy DM with endothelial cells through small corneal incisions.