We examined the ability of fibronectin, an extracellular glycoprotein that interacts with cell surfaces and matrix components, to bind to glomerular basement membrane and the effect of diabetes on this binding. 125I-labeled fibronectin binding to rat glomerular basement membrane (GBM) was dose dependent, related to time and amount of basement membrane, and inhibited by unlabeled fibronectin but not by unrelated proteins. Binding was reduced approximately 60% when GBM was pretreated with collagenase and approximately 24% when pretreated with chondroitinase plus heparinase. Treatment with NaCl had little effect on binding, whereas reduction with beta-mercaptoethanol removed approximately 25% of the bound 125I-fibronectin. Binding to samples prepared from rats with streptozocin-induced diabetes was significantly increased compared with that observed with control preparations at all concentrations of fibronectin and of basement membrane tested. The findings provide direct evidence that fibronectin binds to GBM and that this binding, which represents a biologic function of the protein, is enhanced in diabetes.