Incubation of purified rat glomerular basement membrane (GBM) with [14C]-glucose in vitro resulted in the incorporation of [14C] into acid-precipitable radioactivity in a reaction that was time and temperature dependent. Findings with rat lens capsule basement membrane (LCBM), an anatomically distinct but chemically similar extracellular matrix, incubated for varying times at different temperatures with [14C]-glucose at constant specific activity were similar. Nonenzymatic glycosylation of basement membrane, documented by hydroxymethylfurfuraldehyde generation after incubation with unlabeled glucose, increased in proportion to the ambient glucose concentration over a range of 5--100 mM. Acid-precipitable radioactivity also increased in proportion to [14C]-glucose concentration, although this method overestimated glycosylation about 15-fold at 5--20 mM glucose and 50-fold at 50--100 mM glucose. Coupled with recent in vivo studies, these findings indicate that exposure to increased glucose concentration alters the chemistry of glomerular and other basement membranes. Since accumulation of basement membrane characterizes several of the microangiopathic sequelae of diabetes, the role of increased nonenzymatic glycosylation on the structure, function, and metabolism of basement membrane warrants investigation.