The dorsal root entry zone (DREZ) of the spinal cord is the interface between the central and peripheral nervous systems and is the pathway through which sensory afferents enter the central nervous system during development. However, in the rat, the DREZ becomes a boundary to regenerating sensory axons after Postnatal Days 2-3. The cellular and molecular mechanisms that cause regenerative failure at the DREZ after the critical period for regeneration are unknown. Recent studies demonstrate that two extracellular matrix molecules, Cytotactin/tenascin (CT) and chondroitin 6-sulfate-containing proteoglycans (C-6S-PG) are present in normal boundary regions of the brain and spinal cord during development. In the present study we sought to visualize the expression of these two putative inhibitory molecules in the DREZ of normally developing and adult animals, and also in animals after injury. CT and C-6S-PG spread laterally from the midline to the DREZ by Postnatal Day 3, correlating exactly with the end of the critical period. The staining intensity for these two molecules increases further in the DREZ after root lesions, but not sciatic lesions, at ages when axons cannot regenerate into the spinal cord. Following root lesion CT and C-6S-PG were mostly present in association with reactive glia at the DREZ and in white matter, rather than with reactive glia in grey matter of the dorsal horn, suggesting that astroglia are heterogeneous in their response to root lesion. The coexpression of CT and C-6S-PG may create a molecular barrier which might channel or deflect axons at the DREZ during CNS development and inhibit their growth during regeneration.