The ultrastructures of anionic sites in rat glomerular basement membranes (GBM) were studied at different perfusion pressures of 100, 150, 200 or 250 cm H<sub>2</sub>O by a quick-freezing and deep-etching (QF-DE) method, in addition to conventional fixation methods, using polyethyleneimine (PEI) as a cationic tracer. By the QF-DE method, three-dimensional ultrastructures at each pressure were observed more clearly than those seen on conventional ultrathin sections. When the perfusion pressures were changed from low levels to higher ones, the total thickness of GBM with anionic sites became gradually thinner. Many PEI particles were observed around filaments, not only in the laminae lucidae, but also in the lamina densa. These findings indicated the existence of anionic sites in both laminae lucidae and lamina densa of GBM. The numbers of PEI particles in the lamina rara externa were counted on conventional ultrathin sections for morphometric analyses. The numbers per unit length of GBM were significantly decreased at higher perfusion pressures (200 and 250 cm H<sub>2</sub>O) than those seen at both normal (150 cm H<sub>2</sub>O) and lower (100 cm H<sub>2</sub>O) pressures. It is concluded that the ultrastructures of anionic sites in the GBM may be changed in vivo, depending on the hemodynamics in the glomerular capillary.