Objective: To elucidate the pathogenesis of macular hole formation, focusing in particular on the possible role of cellular migration on the cortical vitreous and internal limiting membrane (ILM) around the macular hole. Methods: To gain a comprehensive overview of the ILM excised in macular hole surgery (n=36), the ILMs were carefully unfolded and spread out onto glass slides as continuous flat sheets that each contained a macular hole. The specimens were observed by light microscopy and transmission electron microscopy (n=9), and the cellular distribution was analyzed by scanning electron microscopy in a quantitative manner (n=27). Immunohistochemistry for glial fibrillary acidic protein and cytokeratin 18 was carried out for cellular characterization. Cellular proliferation was assessed by immunohistochemistry for proliferating cell nuclear antigen and Ki-67. Results: Cellular migration was not apparent around the macular hole in the early stage of development of the macular hole (stage 2, 0 μm). As the macular hole passed through the later stages of development, cellularmigration developed around the macular hole (stage 3, 84 μm) and the area of cellular migration gradually enlarged (stage 4, 420 μm). The immunophenotypic analysis showed that these cells were mainly glial fibrillary acidic protein-positive glial cells and cytokeratin 18-positive retinal pigment epithelial cells. The proliferating cell nuclear antigen and Ki-67 immunohistochemistry showed that some of these cells were proliferating on the ILM. Conclusions: Cellular migration on the ILM is not necessary for the initial formation of a macular break. Cellular migration developed after the macular break occurred, and the migration and proliferation increased gradually from the macular hole. Clinical Relevance: This study provides a new method for understanding the ultrastructural analysis of the pathogenesis of the macular hole. Methods: To gain a comprehensive overview of the ILM excised in macular hole surgery (n = 36), the ILMs were carefully unfolded and spread out onto glass slides as continuous flat sheets that each contained a macular hole. The specimens were observed by light microscopy and transmission electron microscopy (n = 9), and the cellular distribution was analyzed by scanning electron microscopy in a quantitative manner (n = 27). Immunohistochemistry for glial fibrillary acidic protein and cytokeratin 18 was carried out for cellular characterization. Cellular proliferation was assessed by immunohistochemistry for proliferating cell nuclear antigen and Ki-67. : Cellular migration was not apparent around the macular hole in the early stage of development of the macular hole (s tage 2, 0 μm). As the macular hole passed through the later stages of development, cellularmigration developed around the macular hole (stage 3, 84 μm) and the area of ​​cellular migration gradually enlarged (stage 4, 420 μm). The immunophenotypic analysis showed that these cells were mainly glial fibrillary acidic protein-positive glial cells and cytokeratin 18-positive retinal pigment epithelial cells. The proliferating cell nuclear antigen and Ki-67 immunohistochemistry showed that some of these cells were proliferating on the ILM. Conclusions: Cellular migration on the ILM is not necessary for the initial formation of a macular hole. Cellular migration developed after the macular break. and the migration and proliferation increasingly gradually from the macular hole. Clinical Relevance: This study provides a new method for understanding the ultrastructural analysis of the pathogenesis of the macular hole.