Previous studies demonstrated regional differences in the synthesis of extracellular matrix by astrocytes during optic nerve head (ONH) maturation and in glaucomatous optic neuropathy, suggesting heterogeneity of astrocytes. To characterize different types of glial cells in human fetal and adult ONH, we used a variety of neural cell markers such as HNK-1/N-CAM, A2B5, galactocerebroside (GalC), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cryostat or paraffin sections were prepared from fetal (16-25 weeks) and mature (8 months to 75 years old) ONH and processed for standard single/double immunocytochemistry. Two subpopulations of type 7 astrocytes were present in the mature prelaminar and laminar regions. Glial cells expressing only GFAP were identified as type 1A astrocytes at the edges of the cribriform plates. Cells forming the glial columns and lining the cribriform plates expressed both GFAP and HNK-1/N-CAM and were identified as type 1B astrocytes. In the myelinated nerve, type 1A astrocytes form the glial limiting membrane. Cells labeled with GFAP and A2B5 were identified as type 2 astrocytes, and GFAP-negative cells labeled with GalC, MBP, and HNK-1/N-CAM were identified as oligodendrocytes. In fetal ONH, all glial cells expressed HNK-1/N-CAM. In older fetal ONH, some glial cells also expressed GFAP. No type 2 astrocytes or oligodendrocytes were present in the fetal ONH. In conclusion, at least two subpopulations of type 1 astrocytes exist in human ONH: Type 1A astrocytes may serve as structural support for axons; type 1B astrocytes, which retain the developmental neural marker HNK-1/N-CAM, may have a more complex function by interfacing between blood vessels and other connective tissue surfaces. These findings demonstrate the heterogeneity of astrocytes in the human ONH and suggest differential regional responses to changes in their microenvironment.