Tenascin-R, an extracellular matrix constituent expressed by oligodendrocytes and some neuronal cell types, may contribute to the inhibition of axonal regeneration in the adult central nervous system. Here we show that outgrowth of embryonic and adult retinal ganglion cell axons from mouse retinal explants is significantly reduced on homogeneous substrates of tenascin-R or a bacterially expressed tenascin-R fragment comprising the epidermal growth factor-like repeats (EGF-L). When both molecules are presented as a sharp substrate border, regrowing adult axons do not cross into the tenascin-R or EGF-L containing territory. All in vitro experiments were done in the presence of laminin, which strongly promotes growth of embryonic and adult retinal axons, suggesting that tenascin-R and EGF-L actively inhibit axonal growth. Contrary to the disappearance of tenascin-R from the regenerating optic nerve of salamanders (Becker et al., J Neurosci 19: 813–827, 1999), the molecule remains present in the lesioned optic nerve of adult mice at levels similar to those in unlesioned control nerves for at least 63 days post-lesion (the latest time point investigated), as shown by immunoblot analysis and immunohistochemistry. In situ hybridization analysis revealed an increase in the number of cells expressing tenascin-R mRNA in the lesioned nerve. We conclude that, regardless of the developmental stage, growth of retinal ganglion cell axons is inhibited by tenascin-R and we suggest that the continued expression of the protein after an optic nerve crush may contribute to the failure of adult retinal ganglion cells to regenerate their axons in vivo. GLIA 29: 330–346, 2000. 2000 Wiley-Liss, Inc.