In this study a spontaneously formed corneal epithelial cell line, namely HCE-S, was established and characterised. The cell line was karyotyped and corneal epithelial maker expression of the cell line was assessed by immunostaining and semi-quantitative RT-PCR. The morphological characteristics were investigated using SEM and TEM analyses. The functional response to EGF in terms of cell proliferation, wound healing and cell migration was tested using Alamar Blue, scratch wound and colony dispersion assays, respectively. The cells were maintained in culture for more than 100 divisions and 35 passages suggesting that an immortalised cell line had been established. HCE-S, has maintained an epithelial morphology and has not phenotypicaly changed through passages. SEM and TEM microscopy showed morphological similarities to primary corneal epithelial cells. HCE-S expressed a battery of characteristic markers of primary corneal epithelial cells including cytokeratin 3 and PAX 6 as well as the basal cell integrins β1 and α9 and the putative corneal stem cell marker ABCG2. HCE-S cells were responsive to exogenous EGF as shown by proliferation, migration and scratch wound assays. HCE-S can be cultured in a simple DMEM and only serum-based media which gives them an advantage against available corneal epithelial cell lines. This fact, along with the often limited availability and variability of primary corneal epithelial cells and the similarities of the cell line with primary cell characteristics suggest that HCE-S could be a useful tool for the study of corneal epithelial cell biology, ocular surface toxicity studies and pharmacological testing.