Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated 1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of 1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced 1 integrin activation : 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photo-affinity labeling; 2) heparitinase I treatment of cells abrogated 1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) siRNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced 1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of 1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with PKC inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains, and then induced conformational change in 1 integrin necessary for its functional activation. A lateral interaction of 1 integrin with the extracellular region of syndecan-4 molecule may be involved in this conformation change.