The limbal basal epithelium contains the stem cells of the corneal epithelium. In order to study the regulation of these cells we developed models allowing their investigation. While an explant model based on n-heptanol debridement did not allow outgrowth of corneal epithelium onto culture dishes, a serum-free single-cell clonal growth supported colony growth in limbal cells. MCDB 151 medium was supplemented with insulin, transferrin, selenium, hydrocortisone, phospho-/ethanolamine and calcium. Single cells (5,000) were seeded onto 60 mm plastic dishes following combined digestion with dispase and trypsin/EDTA. Increasing CFE was noted until day 6, allowing increasing recruitment into proliferation. This model will serve as a tool for the further investigation of factors that might govern the regulation of the differentiation and proliferation of limbal epithelial cells.