The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 °C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 × 10−7M while the KDfor glycated fibronectin and type IV collagen was 2.9 × 10−6M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 × 10−7M for the interaction of control fibronectin with gelatin vs. KDof 1.2 X 10–6 M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H] heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding. Both a low-affinity (KD= 2 X 10−7M) binding site and a high-affinity [KD= (2–3) × 10−8M] binding site were found for heparin on each of these fibronectin molecules. However, when fibronectin and [3H]heparin were incubated together with type IV collagen at fixed concentrations, the binding of [3H]heparin to fibronectin was significantly enhanced by the addition of type IV collagen. This positive cooperative binding of heparin with fibronectin and type IV collagen was reduced approximately 36% when Fibronectin was nonenzymatically glycated, 64% when type IV collagen was nonenzymatically glycated, and 70% when both Fibronectin and type IV collagen were nonenzymatically glycated (12 days of incubation with excess glucose). The results of the latter binding studies are almost identical with those conducted with fibronectin, heparin, and gelatin [Tarsio, J.F., Wigness, B., Rhode, T.D., Rupp, W.M., Buchwald, H., & Furcht, L.T. (1985) Diabetes 34, 477–484] even though type IV collagen was nonenzymatically glycated to a significantly lesser extent than gelatin. In addition, a slight but significant reduction in the binding of [3H] heparin to Fibronectin and type IV collagen could be observed even with fibronectin nonenzymatically glycated for 1 day in vitro (a level of glycation 3.2-fold that of nonglycated Fibronectin). These observations with native type IV collagen are discussed relevant to changes in the molecular composition of basement membranes that occur in diabetes mellitus.