To compare the effect of mechanical and alcohol-assisted excision on the histological ultrastructure of epithelial disks from human corneas. Vardinoyiannion Eye Institute of Crete, University of Crete, Medical School, Crete, Greece. Ten eyes of 10 patients were deepithelialized by 1 of 2 two techniques. In 6 eyes, a customized instrument was used to mechanically separate the epithelial layer. In 4 eyes, the epithelial disks were obtained using the conventional laser-assisted subepithelial keratectomy (LASEK) technique; that is, with alcohol concentrations of 15% and 20%. All specimens were assessed by light and electron microscopy, and the histological findings of the 2 methods were compared. Transmission electron microscopy showed that when the epithelial disks were excised by mechanical separation, the lamina densa and lamina lucida were preserved and the hemidesmosomes had normal morphology along almost the entire length of the basement membrane. The basal epithelial cells of the separated epithelial disks showed minimal trauma and edema. Specimens obtained using 15% and 20% alcohol concentrations showed formation of cytoplasmic fragments of the basal epithelial cells, enlargement of the intercellular spaces, and extensive discontinuities in the basement membrane, which was excised at the level of the lamina lucida. Mechanical separation did not affect the normal cell morphology of the excised epithelial disks. Transmission electron microscopy of the specimens proved the manual technique is less invasive to epithelial integrity than LASEK using either alcohol concentration.