One of the major complicating factors in insulin-dependent diabetes mellitus is nephropathy. Several investigators have linked heparan sulfate (HS) alterations in the glomerular basement membrane (GBM) with albuminuria as a marker of abnormal blood filtration and the subsequent progression to renal failure. In this study, we examined the fine structure of HS in the glomerulus and the GBM isolated from the kidneys of rats injected with streptozotocin. Using fluorophore-assisted carbohydrate electrophoresis, we obtained disaccharide composition analyses for HS. In a time course study, we observed that normal rat HS isolated from the GBM becomes more N-sulfated as the glomeruli mature over a period of 8 weeks. Diabetic rats injected with streptozotocin at the beginning of this period showed a reversal of this trend. Using a graded sieve technique, we found that two different sizes of glomeruli could be isolated from the rat kidneys and that there was a significant difference in the HS disaccharide content between these two pools of glomeruli. Only the larger sized glomeruli had less N-sulfation of HS as a result of insulin-dependent diabetes mellitus. This change in the fine structure of HS was localized to the GBM and was not associated with cell surface HS. We also generated oligosaccharides of HS that portray fine structural alterations in the diabetic rats indicative of a loss of the sulfation of N-acetylglucosamine.