Tumor necrosis factor‐α (TNF‐α) mediates a range of cellular responses, which have potentially detrimental consequences that affect multiple cell types. To determine whether TNF‐α contributes to glaucomatous optic neuropathy, we have studied the expression of this cytokine and its receptor, tumor necrosis factor receptor‐1 (TNF‐ R1), in human glaucomatous optic nerve heads from patients with different stages of disease using double labeling fluorescence immunohistochemistry. We have also investigated the ability of this cytokine to induce nitric oxide synthase (NOS‐2) in cultured human optic nerve astrocytes by immunocytochemistry and immunoblot. Normal tissue showed constitutive expression of TNF‐R1 in the vasculature of the optic nerve heads but no positive labeling for TNF‐α. In the glaucomatous optic nerve heads, the expression of both TNF‐α and TNF‐R1 were apparently upregulated, primarily in glial fibrillary acidic protein (GFAP)‐positive astrocytes, and appeared to parallel the progression of optic nerve degeneration. In eyes with severe glaucomatous damage, some HLA‐DR positive microglia also contained TNF‐α and TNF‐R1. In the most severely damaged optic nerve heads, the axons of the retinal ganglion cells contained TNF‐R1 and, therefore, are direct targets for neurodegeneration caused by TNF‐α. In vitro astrocytes constitutively express TNF‐R1 and TNF‐α stimulation induces expression of NOS‐2. We hypothesize that TNF‐α contributes to the progression of optic nerve degeneration in glaucoma by both a direct effect on the axons of the retinal ganglion cells and by inducing NOS‐2 in astrocytes. GLIA 32:42–50, 2000. © 2000 Wiley‐Liss, Inc.