The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Götz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell-binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins.