PURPOSE. To develop and characterize a telomeraseimm- ortalized human corneal epithelial cell line (hTCEpi) to serve as an in vitro model for studying the molecular mechanisms involved in regulating human corneal epithelial cell differentiation. METHODS. Primary cultures of human corneal epithelial cells were infected with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Infected hTCEpi cells were selected, cloned, and characterized to identify telomerase activity, proliferative capacity, karyotype, and differentiative potential in routine culture and under consecutive submerged and air- lifted conditions. Cells were evaluated to measure cell cycle kinetics (anti- Ki- 67, anti- p16), stratification (phalloidin and anti- ZO- 1), and differentiation (anti- K3, anti- BCL- 2 and TUNEL labeling). RESULTS. hTCEpi cells exhibited telomerase activity, a normal karyotype and cell cycle kinetics at greater than 240 population doublings, and loss of p16 after passage 10. Air- lifting produced a well stratified epithelium (five to seven cell layers) with apical ZO- 1- stained tight junctions. Submersed culture demonstrated increasing expression of stratification markers (K5/K14) with K3- corneal keratin marker expression in longterm, air- lifted culture. Anti- BCL- 2 staining showed both nuclear and cytoplasmic localization with loss of nuclear BCL- 2 expression in TUNEL- labeled surface epithelial cells. CONCLUSIONS. hTCEpi cells stratify, differentiate, and desquamate similar to normal human corneal epithelium. Further study of the hTCEpi cell line may be valuable in studying the molecularmechanisms regulating corneal epithelial cell differentiation and desquamation. PURPOSE. To develop and characterize a telomeraseimm- ortalized human corneal epithelial cell line (hTCEpi) to serve as an in vitro model for studying the molecular mechanisms of in human corneal epithelial cell differentiation. METHODS. Primary cultures of human corneal epithelial cells were infected with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Infected hTCEpi cells were selected, cloned, and characterized to identify telomerase activity, proliferative capacity, karyotype, and differentiative potential in routine culture and under consecutive submerged and air-lifted conditions. Cells were evaluated to measure cell cycle kinetics (anti- Ki- 67, anti-p16), stratification (phalloidin and anti- ZO- 1), and differentiation (anti- K3, anti- BCL- 2 and TUNEL labeling). RESULTS. hTCEpi cells exhibited telomerase activity, a normal karyotype and cell cycle kinetics at greater than 240 population doublings, and loss of p16 after passage 1 0. Air-lifting produced a well stratified epithelium (five to seven cell layers) with apical ZO- 1-stained tight junctions. Submersed culture demonstrated increasing expression of stratification markers (K5 / K14) with K3-corneal keratin marker expression in longterm, Anti- BCL- 2 staining showed both nuclear and cytoplasmic localization with loss of nuclear BCL- 2 expression in TUNEL-labeled surface epithelial cells. CONCLUSIONS. hTCEpi cells stratify, differentiate, and desquamate similar to normal human corneal epithelium. Further study of the hTCEpi cell line may be valuable in studying the molecular mechanisms of regulating corneal epithelial cell differentiation and desquamation.