Endothelial cells were obtained from 23 human corneal-scleral rims. Donors were 0.2-37 yr of age. Most cultures obtained from donors under 30 yr ceased to grow after the seventh passage (3-24 weeks). Growth from 17- and 25-yr-old donors were maintained for nine passages (26 weeks) and 18 passages (100 weeks), respectively. Cultures were established from 12 rims, using growth factors in uncoated culture flasks or epidermal growth factor (EGF) in an extracellular matrix-coated (ECM) flask. EGF improved the growth potential of the cells; a combination of an ECM-coated-tissue culture flask with EGF in the medium provided an improved environment for continuous growth.