Purpose: To modify the isolation of human retinal pigment epithelial (RPE) cells and to increase the purification and production of cultured RPE cells.Methods: The human eyecups were fixed on a rubber holder. After digestion by trypsin, RPE cells were collected, then cultured and identified by morphology, im-munohistochemistry and electron microscopy.Results: The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm. These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining. Cellular microvilli and tight junctions could be seen through transmission electron microscopy. Conclusion: We developed a rubber holder to fix the eyecup. Using this holder, more and purer cultured RPE cells can be obtained. These cultured RPE cells are similar to those in vivo in morphology and immunohistochemical staining. Eye Science 1999; 15: 187 - 190. Purification of human retinal pigment epithelial (RPE) cells and to increase the purification and production of cultured RPE cells. Methods: The human eyecups were fixed on a rubber holder. After digestion by trypsin, RPE cells were collected, then cultured and identified by morphology, im-munohistochemistry and electron microscopy. Results: The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm. These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining Cellular microvilli and tight junctions could be seen through transmission electron microscopy. We developed a rubber holder to fix the eyecup. Using this holder, more and purer cultured RPE cells can be obtained. in morphology and immunohistochemical staining. Eye Science 1999; 15: 187-190.