To review recently published experimental data on human corneal endothelium (CE) precursors. A sphere-forming assay was used for the isolation of human CE precursors from human donor corneas and cultured human CE. CE morphology derived from precursors was compared with that from cultured human CE. Human CE from donor corneas formed primary and secondary sphere colonies and expressed neural and mesenchymal proteins. The progeny of these colonies had a human-CE-like hexagonal shape and showed transport activity, suggesting that the isolated spheres were indeed precursors of human CE. Similar precursors were isolated from cultured human CE, which provided further evidence that the sphere-forming assay facilitates the mass production of human CE precursors. Cultured human CE obtained in this manner had a regular hexagonal morphology in contrast to passaged, cultured human CE. The sphere-forming assay may become a powerful tool for the regeneration of human CE by precursor injection and the construction of cultured CE sheets.