To better define the growth requirements for corneal epithelial cells, methods for serumfree culture were established. Sheets of corneal epithelium from eyes of swine and rabbits were obtained by dispase treatment of corneal buttons, and single cells and small clumps were obtained by further dissociation with trypsin/EDTA. Growth of cells with epithelial-like morphology was readily achieved in low calcium MCDB 153 medium containing epidermal growth factor, insulin, hydrocortisone, and bovine pituitary extract. Primary cultures could be subcultivated at least four times at 1:6 split ratios. Examination of cultured corneal epithelial cells by transmission electron microscopy demonstrated a relatively undifferentiated phenotype. Elevation of calcium in the medium caused the reappearance of desmosomal junctions and a more typical corneal epithelial morphology.