Isolated rat renal glomeruli incorporate radioactive sulfate into glycosaminoglycans, which are integral components of the glomerular basement membrane. Cellulose acetate electrophoresis and specific enzymatic sensitivities of glycosaminoglycans prepared after pronase digestion of purified glomerular basement membrane indicate the presence of heparan sulfate. We examined the effect of experimental diabetes on the incorporation of [(35)S]-sulfate into glycosaminoglycans deposited into newly synthesized glomerular basement membrane in vitro. Basement membranes were purified from glomeruli isolated from normal and streptozotocin-diabetic rats after incubation for 2 hr with radiolabeled sulfate and then were subjected to pronase digestion for isolation of the glycosaminoglycans. [(35)S] incorporation into basement membrane glycosaminoglycans was significantly decreased in glomeruli from diabetic animals. The addition of insulin (100 micron U/ml) in vitro did not affect [(35)S] incorporation into glycosaminoglycans of the glomerular basement membranes in normal or diabetic glomeruli. High glucose concentration (5 vs. 20 mM) was without effect in short-term incubations of glomeruli from normal animals. The results indicate that experimental diabetes influences [(35)S] sulfate incorporation into glomerular basement membrane glycosaminoglycans and suggest that decreased heparan sulfate production and/or sulfation may contribute to the increased permeability of the glomerular basement membrane in diabetes.