Fibulin is a recently described extracellular matrix (ECM) and plasma glycoprotein (Argraves, W. S., Tran, H., Burgess, W. H., and Dickerson, K. (1990) J. Cell Biol. 111, 3155-3164). In this report, ligand affinity chromatography and solid-phase binding analyses were performed to determine which ECM protein(s) interact with fibulin. Fibulin-Sepharose bound two polypeptides of 240 and 100 kDa from the culture medium of metabolically radiolabeled fibroblasts. These two proteins were identified as fibronectin (FN) and fibulin, respectively, based on their electrophoretic behavior and reactivity with monoclonal antibodies. Consistent with the findings of affinity chromatography, fibulin bound to surfaces coated with FN (either plasma or cellular form) or fibulin but not with other ECM proteins, such as laminin, merosin, and types I and IV collagen. The binding of fibulin to solid-phase FN was estimated to have a Kd of 139 nM, whereas the Kd for self-interaction was 322 nM. Evaluation of proteolytic fragments from all regions of FN allowed a fibulin-binding site to be localized within a 23-kDa heparin-binding fragment containing type III repeats 13-14. Heparin did not compete for the interaction between fibulin and FN, suggesting that the binding sites for fibulin and heparin are distinct.