Many extracellular matrix glycoproteins--including laminin, fibronectin, thrombospondin, type I collagen, and other collagens--bind the glycosaminoglycan heparin, yet little is known about the functional significance of these interactions. It is also not known if heparin-binding extracellular matrix proteins recognize distinct structural elements in heparin, nor whether all extracellular matrix proteins recognize the same or different aspects of heparin structure. If extracellular matrix proteins each recognize distinct features of heparin, such specificity could be of importance in vivo, where structurally distinct heparan sulfate species occur. To investigate specificity in the binding between extracellular matrix proteins and heparin, the method of affinity coelectrophoresis (ACE) was used [Lee, M. K., & Lander, A. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2768-2772]. Low M(r) (approximately 6 kDa) 125I-heparin was fractionated by electrophoresis through agarose gel lanes containing extracellular matrix proteins at various concentrations; from heparin migration patterns, binding affinities were calculated. The results indicate that fibronectin, type I collagen, and laminin--but not thrombospondin--each fractionate heparin into subpopulations that differ substantially in binding affinity. From ACE gels containing either fibronectin, type I collagen, or laminin, fractions of heparin were isolated that represent the 25% of molecules most strongly bound and the 25% least strongly bound by each of these proteins. Subsequent ACE analysis of these six fractions showed that (1) for each of fibronectin, type I collagen, and laminin, strongly- and weakly-binding heparin subfractions differ approximately 5-30-fold in Kd; (2) heparin that binds strongly to any one of fibronectin, type I collagen, or laminin also binds strongly to the other two; (3) heparin that binds weakly to any one of fibronectin, type I collagen, or laminin, also binds weakly to the other two; (4) heparin subfractions that differ greatly in affinity for fibronectin, type I collagen, and laminin show little difference in Kd for thrombospondin or for the heparin-binding growth factor basic fibroblast growth factor (bFGF); (5) neither heterogeneity in molecular charge [as measured by diethylaminoethyl (DEAE) chromatography] nor size nor the presence or absence of antithrombin III recognition sequences can account for the selective binding of heparin subpopulations to fibronectin, type I collagen, and laminin. These results suggest that structural elements within heparin can confer preferential binding to extracellular matrix proteins. Sensitivity of some, but not all, extracellular matrix proteins to these structural features suggests that similar features, if present in heparan sulfates or other glycosaminoglycans, may be physiologically relevant in vivo.