Purpose We sought to clarify the morphologic features of macular hole development. Methods Using scanning laser ophthalmoscopy, we examined 47 eyes with macular holes before and after vitrectomy. Cases included three eyes with stage IB disease (foveal detachment), 14 with stage 2 (break at the fovea), 21 with stage 3 (full-thickness macular hole), and nine with stage 4 (detached operculum), according to Gass's classification. Ten eyes with central serous chorioretinopathy served as controls. Results Radiating striae of Henle's fibers were seen around the elevated rim of the macular cyst or hole when the intraretinal structures were illuminated by helium-neon laser. Granularity was observed on the macular hole floor. A granular reflex corresponding to the macular break or hole, but no radiating striae were seen by argon blue laser. In 40 eyes in which macular holes were successfully closed by vitrectomy, radiating striae of Henle's fiber layer disappeared, and the normal foveal depression was restored. The ten eyes with central serous chorioretinopathy had a normal foveolar depression and no detectable radiating striae of Henle's fiber layer, despite full-thickness retinal detachment in the macular area. Conclusions Tractional elevation of Henle's fiber layer with intraretinal foveolar cyst formation is the initial feature of macular hole development. A macular hole is formed when the anterior cyst wall containing Henle's fiber layer is operculated. Remnants of the photoreceptor cell layer remain on the hole floor in the early stage of macular hole development and subsequently degenerate.