The ultrastructure of glial cells and extracellular matrix that support normal guinea pig optic nerve axons at the lamina cribrosa was examined by conventional thin-section transmission electron microscopy and by the quick-freeze, deep-etch, rotary-shadow technique. Glial cell bodies and processes were identified by their characteristic parallel intermediate filaments that were closely packed with reduced cross-linking between adjacent filaments when compared with neighboring axons. Glial processes surrounded lamina cribrosa beams and separated axons from the beams. These beams contained bundles of collagen fibrils. Short regular cross-bridges spanned the spaces between the collagen fibrils much like rungs of a ladder. Surrounding the collagen bundles was a network-like peribeam extracellular matrix. This matrix extended to the basement membrane of the surrounding, stratified processes of glial cells. The glial basement membrane was connected to the glial plasma membrane by numerous orthogonal fibrous strands. In regions within basement membrane there also were cross-bridges from the glial plasma membrane to intermediate filaments within the cell. Apposed glial process membranes appeared to directly contact each other. Unmyelinated optic nerve axon membranes and glial process membranes also directly abutted each other. Overall, the cross-bridging structural organization was dense near collagen beams, and became progressively less dense toward the axon. This organization of the lamina cribrosa may play an important role in the distribution of tissue shear stress associated with intraocular pressure and thus protect the retinal ganglion cell axons as they pass through the lamina cribrosa.