AIM: To examine the morphological characteristics and antigen expression patterns of cultured human retinal glia to define novel subtypes. METHODS: Morphologic characteristics and marker expression were examined during cultivation using hematoxylin and eosin (HE) and immunostaining for glial fibrillary acidic protein (GFAP) and vimentin. RESULTS: A subtype of human retinal glia distinct from radial glia (Muller cells) was successfully isolated by digesting the retina first in diastase vera (pancreatin) and then in clostridiopeptidase, followed by culture on fibronectin substrate in human endothelial cell medium (supplemented with 10% fetal bovine serum, growth factors, and heparin sodium). Adherence was detected at 72h and cell-cell coupling at 9d-10d after seeding. These cells were extensively and strongly immunopositive for GFAP and vimentin, consistent with glial expression patterns in the human retina, but were morphologically and immunohistochemically distinct from previously reported cultured retinal glia, including GFAP -positive and glutamine synthetase (GS)-positive Muller cells. CONCLUSION: A unique human retinal glial cell type can be isolated using diastase vera and clostridiopeptidase and then maintained vitra Further studies are required to characterize the physiological and pathological functions of these cells.