The keratocytes are specialized mesenchymal cells that produce and maintain the extracellular matrix of the corneal stroma. With a typical dendritic and flattened appearance, these cells can morph into fibroblasts and myofibroblasts upon injury, and produce abnormal or fibrotic extracellular matrices detrimental to corneal transparency. Insights into mechanisms that regulate these phenotypic switches and optimal culture conditions that preserve the keratocyte phenotype are important for tissue engineering of the corneal stroma. Like other cell types with self-renewing capacity, keratocytes can form spheres in culture. Here we investigated human and bovine keratocytes with respect to their sphere forming capabilities, and sought to identify potentially distinguishing markers for the keratocyte and fibroblast phenotypes. Keratocytes, isolated from bovine and human corneas, cultured in serum-free medium supplemented with insulin, selenium and transferrin, assumed typical keratocyte morphology, converted to fibroblasts in serum-containing medium and reverted to keratocytes after serum-deprivation. The bovine keratocytes produced spheres under adherent or low attachment conditions, while the human keratocytes produced spheres under low attachment conditions only. The primary keratocytes and fibroblasts expressed vimentin, confirming their mesenchymal origin. Keratocan, considered to be a marker for keratocytes, was also detected in early passage bovine fibroblasts. BMP3 was expressed in keratocytes and keratocyte-derived spheres, while cadherin 5 in keratocytes only, suggesting these as potential keratocyte markers.