Background/Aims: Glomerular basement membranes (GBM) and tubular basement membranes (TBM) consist of a fine meshwork composed mainly of type IV collagen. Each segment of tubules has specialized physiologic functions, and thus we investigated the ultrastructure of various basement membranes in rat kidneys. Methods: Since purifying basement membranes from different tubule segments is technically challenging, we employed tissue negative staining rather than conventional negative staining to compare the ultrastructures of proximal and distal TBM and GBM in normal rats. We also assessed the distribution of extracellular matrix components including type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes by immunohistochemistry. Results: TBM and GBM of normal rats showed a fine meshwork structure consisting of fibrils forming small round to oval pores. Short- and long-pore diameters in proximal tubules were 3.3 ± 0.5 and 3.9 ± 0.6 nm, respectively, and in distal tubules 3.5 ± 0.7 and 4.3 ± 0.8 nm, respectively. For GBM the respective diameters were 2.5 ± 0.5 and 3.0 ± 0.5 nm. Immunohistochemical analysis showed no significant difference in distribution of extracellular matrix components between proximal and distal TBM. However, immunofluorescence scores of α1 chain of type IV collagen, fibronectin, and laminin were higher in the TBM than in the GBM. On the other hand, heparan sulfate proteoglycan was higher in the GBM. Conclusion: Ultrastructural differences in renal basement membranes may be related to differences in physiologic function in each segment.