This study reports the nonenzymatic glycation of plasma fibronectin in vivo in diabetic dogs and also in vitro by incubation of human plasma fibronectin with excess glucose. Although no difference is observed in the total plasma fibronectin level, the nonenzymatic glycation of fibronectin is increased 2.3-fold in inbred male beagle dogs made diabetic with alloxan in comparison with age-matched controls. The extent of nonenzymatic glycation of fibronectin is shown to be proportional to blood glucose levels. HPLC reverse-phase analysis of the hydrolized amino acids and glycoamino acids from plasma fibronectin samples of normal and diabetic dogs show that nonenzymatic glycation occurs only on lysine residues. When purified human plasma fibronectin was incubated in vitro with 500 mM glucose, the extent of nonenzymatic glycation of fibronectin was observed to increase proportionately with time. Ligand binding assays conducted in solution with varying concentrations of of 3H-heparin in the presence of a constant amount of normal or nonenzymatically glycated human plasma fibronectin gave virtually identical binding curves. However, the binding of 3H-heparin to normal fibronectin could be increased fourfold by the concomitant addition of normal gelatin (denatured calfskin collagen). If in vitro glycated fibronectin and/or in vitro glycated gelatin are added under this latter condition with 3H-heparin, there is a tremendous decrease in the expected heparin binding seen with normal levels of nonenzymatic glycation. Other experiments were performed to quantitate the binding of 3H-labeled fibronectin to gelatin-coated nitrocellulose filters. Nonenzymatic glycation of fibronectin in vitro resulted in markedly decreased binding of 3H-fibronectin to collagen. Enhanced nonenzymatic glycation of fibronectin in vivo and alterations in the molecular association of fibronectin, gelatin, and heparin brought about by nonenzymatic glycation are discussed relevant to the pathophysiology of the complications of diabetes.