The ultrastructure of the glomerular basement membrane of the long-term diabetic and age-matched control rats was studied with the application of advanced high-resolution microscopy. By using the freeze substitution method for the preparation of the renal tissue, it was possible to observe that the glomerular basement membrane in control and diabetic animals is composed on only a single lamina densa without the presence of a lamina lucida interna or externa. High-resolution electron microscopy of the diabetic glomerular basement membrane showed significant alterations in its morphology and ultrastructure. First, the basement membrane in diabetic condition appeared to be split into two halves, endothelial and epithelial. In the epithelial half of the membrane, the network of distinct strands referred to as cords, which were clearly present in the glomerular basement membrane of age-matched control animals, became less distinct and showed a diffused appearance being evenly replaced by thin filaments. The openings of the network were filled with a granular material. In the endothelial half of the membrane, on the other hand, the cord network was variably lost in diabetic condition and, within the resulting vacant spaces, bundles of fibrils 12 nm in width, identified as basotubules, were deposited. Immunolabeling for type IV collagen was found to be enriched in the endothelial half of the basement membrane being associated with the bundles of basotubules. The ultrastructural changes reported by high-resolution microscopy could be related to the molecular alterations of the basement membrane components and to the loss in permselectivity occurring during diabetes.