We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.