Cytotactin/tenascin (CT/TN) is an extracellular matrix protein that binds to a variety of cell types and that influences neurite outgrowth. It has a multidomain structure with regions homologous to epidermal growth factor (EGF)-like repeats, fibronectin (FN) type II repeats, and the beta and gamma chains of fibrinogen (fg). The current study demonstrates that a fusion protein corresponding to the sixth fibronectin type III repeat in CT/TN (CTfn6) supported cell attachment and promoted an increase in the number of cells with neurites in both central and peripheral neurons in tissue culture. The third fibronectin type III repeat, CTfn3, like intact CT/TN, supported attachment of peripheral neurons but not of central neurons and, while it caused an increase in neurite length, it did not increase the number of cells that sprouted neurites. When CTfn3 and CTfn6 were combined, an increase in both the number of cells sprouting neurites and in neurite length was observed for peripheral neurons that resembled their response to intact CT/TN. Cell attachment to CTfn6 was inhibited in the presence of function-blocking antibodies against beta 1 integrins. In contrast, the interaction with CTfn3 was not inhibited by antibodies to beta 1 integrins, but was inhibited by RGD-containing peptides. The results suggest that cell binding to CT/TN involves two different sites within the molecule and occurs via different receptors which may be differentially expressed on different neuronal cell types. The location of these sites within the whole molecule in the context of other adhesive and counteradhesive domains may modulate their influence on cellular responses such as cell attachment and neurite outgrowth.