Cellular responses to fibronectin (FN) are likely to have a complex molecular basis involving the interactions between multiple functional domains of FN and specific cell surface molecules. We have utilized several types of recombinant FN fragments and their chimeric fragments to examine the regulatory mechanisms of the spreading and chemotactic migration of HT1080 human fibrosarcoma cells on FN. The CH-271 fusion fragment, in which the cell-binding domain (C-274) of FN is adjacent to the heparin-binding domain (H-271), promoted cell spreading more efficiently than C-274, H-271, or their mixture (C-274 + H-271) over a 60-min incubation. The CH-271 variants containing various modules in the heparin-binding domain (CHV peptide) showed different promotion of cell migration, spreading, and vinculin accumulation at focal adhesion, respectively. The preincubation of the cells with heparitinase I resulted in significant inhibition of chemotactic migration to FN and its fragments containing the III13 and/or III14 modules of the heparin-binding domain. Additionally, migration to CH-271 was inhibited by the presence of the RGDS peptide in a concentration-dependent fashion. Thus, the spread and migration responses of HT1080 cells onto FN fusion peptides require the adjacent coexistence of cell- and heparin-binding domains and are mediated by the interactions between cell surface heparan sulfate and the heparin-binding domain, in concert with the interaction between cell surface integrin and the cell-binding domain. In conclusion, our present study demonstrated that fusion peptides of fibronectin can efficiently induce two signals from the cell-binding and heparin-binding domains required for the completion of cell spreading, the formation of focal contact, and motility at the early stage of the culture, suggesting that the III13 or III14 modules within the heparin-binding domain are responsible for the initiation of different cellular responses.