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To define the architecture and extracellular matrix composition of the lamina cribrosa in rodents, normal, adult pigmented rat and guinea pig eyes were frozen and sectioned for light microscopic immunohistochemistry. Antibodies specific for collagens I, III, IV and VI, laminin, elastin, and chondroitin and dermatan sulfate proteoglycans were exposed to longitudinal and cross-sections of optic nerve heads and their binding distributions observed with the avidin-biotin-peroxidase complex technique. Cross-sections of the intraocular portion of the rat optic nerve head revealed a horizontally oval shape with distinct, vertically oriented, laminar beams. The guinea pig optic nervehead cross-section was circular, with randomly oriented beams. In both animals, collagens I, III and VI were found throughout the laminar beams, along with elastin fibrils. Collagen IV and laminin antibodies deposited along laminar beam margins and within the beams, representing astrocytic and vascular endothelial cell basement membranes. Both animals showed evidence for dermatan and chondroitin sulfate-containing proteoglycans in all connective tissue structures of the nerve head. In the rat, chondroitin-4 sulfate proteoglycans appeared localized to the sclera and laminar beams. The rat and the guinea pig optic nerve head possess an identifiable lamina cribrosa with structural proteins nearly identical to that of the primate. Both animals may provide affordable alternative animal models for in vivo studies on the role of the lamina cribrosa in glaucomatous optic nerve damage.