Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.