The corneal endothelium forms a physical barrier that limits the free flow of aqueous humor towards the stroma, which is essential to maintain corneal transparency. Since the regenerative capacity of the endothelium is limited once the monolayer is formed, cell damage and loss of cells with age is only compensated by cell spreading. To date there have been no reports describing effects of age on morphologic appearance bovine corneal endothelial cells (BCEC). In vivo studies of corneal endothelial cells have shown a decrease in cell density and an increase in pleomorphism and polymegethism with advancing age. We characterized size and morphology of BCEC compared to bovine corneal epithelial cells (BCEpC) with age and with time in culture. BCEC and BCEpC were cultured for 8 to 30 days. Cell surface area and size were measured by confocal microscopy and FACS. Immunocytochemistry was used to stain actin cytoskeleton (phalloidin) and microtubules (a-tubulin) and with antibody against von Willebrand factor. To examine cell morphology in vivo, fresh bovine corneas of different ages were processed for scanning electron microscopy (SEM). Cultured BCEC showed a significant increase and in cell size from 710.6  7.8 mm² (n = 3350) in cells on day 8 to 2517.4  67.1 mm² (n = 2454) on day 30 after isolation. In cells cultured for a longer period the monolayer contained larger intercellular spaces and the cells form interdigitations. The changes were not due to variability in corneas, since the increase in cell size with time in culture is also present in BCEC isolated from the same cornea. FACS-data were in line with these findings. In contrast to these changes in BCEC, BCEpC did not show comparable increases in cell size with increasing duration of cell culture. SEM pictures of corneas from different ages showed a difference in cell size. The monolayer from the older cornea showed cell-free areas and the cells were bigger. In conclusion, cultured BCEC show an increase in surface area/size with time in culture similar to the effects of aging. This is likely to affect intercellular communication and barrier properties.