Binding interactions among fibronectin (FN), glycosaminoglycan chains (GAG) and hyaluronate binding proteins (HABP) have been examined using a bead aggregation assay. 3H-FN binds to Dowex beads coated with chondroitin sulfate GAG chains (CS) but not to beads coated with hyaluronic acid (HA). Assays of bead agglutination indicate that FN has multiple binding sites for CS. Binding of 3H-FN to HA-beads is slightly promoted by exogenous divalent cations but this interaction does not aggregate Dowex-HA. The interaction between FN and CS reduces the previously reported ability of Dowex-CS to associate with HA-beads (Turley, E.A. and Roth, S. 1980, Nature Vol. 283, 268-271). HABP bind to HA and to a lesser extent CS. These proteins both stabilize the interaction between HA- and CS-beads in a manner reminiscent of the stabilization of HA-proteoglycan interactions by link protein and permit aggregation of Dowex-CS-FN with Dowex-HA. This simple bead aggregation assay allows rapid characterization of properties that may help to clarify how macromolecular constituents spontaneously assemble into an interstitial matrix.