We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not ( J. Biol. Chem. 264, 8012-8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chon-droitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.