PURPOSE: To develop an in vitro model of human corneal epithelium that can be propagated in serum-free medium that is tissue specific, species specific, and continuously available. METHODS: Primary explant cultures from human cadaver donor corneas were generated and subsequently infected with Adeno 12-SV40 (Ad12-SV40) hybrid virus or transfected with plasmid RSV-T. RESULTS: Several lines of human corneal epithelial cells with extended life span were developed and characterized. Propagation of both primary cultures and lines with extended life span, upon collagen membranes at an air-liquid interface, promoted multilayering, more closely approximating the morphology observed in situ. CONCLUSIONS: In vitro models, using primary cultures of corneal epithelium and lines of corneal epithelial cells with extended life span, retain a variety of phenotypic characteristics and may be used as an adjunct to ocular toxicology studies and as a tool to investigate corneal epithelial cell biology.