Neurites are believed to be guided by astrocyte boundaries during development. We have previously shown that in vitro astrocyte boundaries can be generated by combining two different astrocyte cell lines, one which is inhibitory to neurite outgrowth (Neu7) with one that is permissive (A7). The extracellular matrix molecules tenascin-C, chondroitin sulfate proteoglycans (CSPG) and keratan sulfate proteoglycans (KSPG) were implicated in boundary formation. We have now further addressed the roles of these molecules using additional astrocyte cell lines that differ in their potential to permit neurite extension and in their expression of extracellular matrix molecules. T34-2 and 27A1 cells are permissive to neurite extension. T34-2 cells express high amounts of tenascin-C, but very low levels of proteoglycans, while 27A1 cells express CSPG and KSPG, but very little tenascin-C. T34-2 cells formed boundaries to neurites, and these boundaries are greatly reduced in the presence of blocking antitenascin-C antiserum. The addition of the antiserum did not affect neurite extension. 27A1 cells also formed boundaries without affecting neurite extension. Chondroitinase ABC, but not keratanase, treatment reduced the boundary, suggesting that CSPG is a major boundary component. These results demonstrate that astrocyte tenascin-C and proteoglycans are distinct components of astrocyte boundaries. More importantly, these results suggest that growing neurites can be directed to their targets by astrocyte-derived guidance molecules independent of effects on process extension.