We performed electron immunocytochemical staining for cytokeratins and glial fibrillary acidic protein on cortical vitreous obtained at the time of vitrectomy from two patients with premacular hole lesions. The specimens were thought to represent cortical vitreous because each consisted of a sheet of tissue that, in addition to being firmly attached around the foveal lesion, was attached around the disc and extended well into the periphery. The specimens contained a moderate number of cells and an abundant extracellular matrix. Most of the cells were found singly or in small clusters on the surface of the matrix. Preembedding immunostaining on one specimen showed several cells that stained for cytokeratins and several that stained for glial fibrillary acidic protein. The majority of the cells on the matrix appeared to express one of these two intermediate-filament proteins. Postembedding immunogold double labeling on both specimens showed that most cells were labeled for either glial fibrillary acidic protein or cytokeratins, but there were a few cells that unequivocally expressed both proteins simultaneously. These data suggest that the cortical vitreous of patients with some premacular hole lesions contains retinal pigment epithelial and glial cells that may contribute to abnormal vitreoretinal adherence and could play a role in macular hole formation.