Syndecan-2 cooperates with integrin alpha 5 beta 1 in cell adhesion to a fibronectin substratum and regulates actin cytoskeletal organization in an expression level-dependent manner; Lewis lung carcinoma-derived P29 cells with high expression form stress fibers, whereas the same tumor-derived low expressers, LM66-H11 cells, form cortex actin [Munesue, S., Kusano, Y., Oguri, K., Itano, N., Yoshitomi, Y., Nakanishi, H., Yamashina, I., and Okayama, M. (2002) BIOCHEM: J. 363, 201-209]. In this study we examined the participation of other cell surface heparan sulfate proteoglycans in this signaling. The two clones expressed syndecan-1, -2 and -4, and glypican-1 at similar levels except for syndecan-2. Treatment of cells with phosphatidylinositol-specific phospholipase C or immobilized anti-syndecan-1 antibodies demonstrated that neither glypican-1 nor syndecan-1 was involved in this signaling, indicating that individual cell surface heparan sulfate proteoglycans have functional specificity. Stimulation with immobilized anti-syndecan-2 or -4 antibodies induced stress fiber formation in P29 cells but not in LM66-H11 cells, despite the similar levels of syndecan-4 expression, suggesting that stress fiber formation required a threshold expression level of syndecan-2 acting downstream of syndecan-4. This was confirmed by cells in which syndecan-2 expression was artificially suppressed by antisense mRNA oligonucleotide treatment or elevated by cDNA transfection. This is the first report demonstrating that syndecan-2 and -4 cooperate in situ in actin cytoskeletal organization.