We compared the ultrastructure of the inner limiting membrane (ILM) and epiretinal tissue in closed and non-closed, idiopathic macular holes (MH). Peeling of ILM and epimacular tissue during vitrectomy was successfully performed on 77 eyes with stage III MH and on 19 eyes with stage IV MH. In 16 additional eyes with non-closed MH, we performed a second vitrectomy with extended ILM removal. The specimens were processed for transmission electron microscopy. Fibrocellular proliferation at the vitreal side of the ILM was found in 57% of MH that were closed by one operation, and in 100% of non-closed MH. Mono- and multilayered cellular membranes as well as native vitreous collagen at the ILM were significantly more frequent in eyes with stage IV MH than in eyes with stage III MH. In non-closed MH, cellular proliferation was mostly seen as irregular cell accumulation, and masses of newly formed collagen were found. Since ILM remnants and collagen represent a stimulus for the early formation of tangential traction preventing successful MH closure, it appears mandatory to create a complete vitreoretinal separation and to remove the ILM and collagen thoroughly during MH surgery.