To test the hypothesis that increased nonenzymatic glycosylation contributes to the microangiopathic sequelae of diabetes, we examined the interaction of glucose with basement membranes in vivo and in vitro. The level of nonenzymatic glycosylation, determined by hydroxymethylfurfuraldehyde (HMF) generation, in glomerular basement membrane (GMB) collagen purified from rats with streptozotocin diabetes was significantly greater than that of nondiabetic GMB. Incubation of purified rat GBM and lens capsule basement membrane with [¹⁴C]-glucose in vitro resulted in the incorporation of [¹⁴C] into acid precipitable radioactivity in a reaction that was dependent on time, temperature, and glucose concentration. Glucose-dependent nonenzymatic glycosylation of GBM in vitro was documented by measurement of HMF generation after incubation with unlabeled glucose. These results provide direct experimental evidence that hyperglycemia, via nonenzymatic glycosylation, modifies the chemistry of proteins specifically involved in diabetic glomerulosclerosis.