FVs have been described, depending on mammalian species, as being either fusiform or discoidal in cross-section. According to Staehelin et al., they have a form of biconvex discs with a diameter 0.5–1 mm. Minsky and Chlapowsky proposed that FVs are pancake-like flattened spheres, but this has never been confirmed by ultrastructural 3D analyses. FVs are lined by an asymmetric unit membrane, which contains four major integral proteins, uroplakins Ia, Ib, II and IIIa. Uroplakins form 16-nm intramembranous uroplakin particles, which are hexagonally arranged in urothelial plaques. Plaques measure between 0.3 and 1 mm in diameter, and they are connected by a non-thickened membrane, called hinge region. UPs are synthesized in the endoplasmic reticulum, where UPIa and UPIb form heterodimers with UPII and UPIIIa, respectively. Conformational changes in the Golgi apparatus enable the formation of 16-nm intramembranous particles, which are hexagonally arranged into 2D crystalline plaques in the post-Golgi compartments. While the structure of the 16-nm particles is largely known, the information on the 3D structure of mature FVs is missing. The plaque composition of mature FVs is identical to that of the apical plasma membrane of umbrella cells, therefore it has been proposed that FVs are transported from the Golgi apparatus towards the apical cell surface where they fuse with the plasma membrane. According to one hypothesis,Timosaponin-BII FVs are inserted into the apical plasma membrane during bladder distension and retrieved during bladder contraction. This membrane recycling therefore provides a mechanism to adjust surface area of umbrella cells during distension-contraction cycles of the urinary bladder. Alternative hypothesis says that FVs are not retrieved during contraction of the bladder; instead the apical surface area is accommodated only by the apical plasma membrane infolding. The analyses of morpho-functional organization of FVs are therefore essential for understanding their role in the intracellular membrane traffic and in the turn-over of the apical plasma membrane. Electron tomography, which allows 3D reconstructions of objects with the resolution below 10 nm, has greatly contributed to the understanding of subcellular structures and compartments. In order to analyse subcellular structures by ET in the state ‘close to native’, samples should be fixed by high pressure freezing, which allows immobilization within milliseconds,Theaflavin followed by freeze substitution. Because FVs are relatively large compartments, their 3D reconstruction requires serial sectioning and joining of tomograms. Here we demonstrate that high pressure freezing and ET of serial sections, supported by freeze-fracture and immunocytochemistry, give a new insight into the structure and organization of FVs in umbrella cells. We also compared the arrangement of FVs during distension-contraction cycle of the urinary bladder, and analysed intermediate cells with respect to the occurrence of FVs. FVs are highly specialized compartments that transport urothelial plaques in umbrella cells of the urothelium. Here we employed ET, freeze-fracturing and immuno-electron microscopy of the mouse urothelium in order to resolve the 3D ultrastructure and organization of FVs in their ‘close to native’ state. Our results indicate that rapid cryo-fixation is the superior method for ultrastructural studies of FVs and their morphofunctional organization in umbrella cells. Until now, conclusions about the nature of FVs were based on observations of ultrathin sections and freeze-fracturing whereas 3D models were not done yet. To investigate cellular structures in 3D rather than their 2D projections, ET is the method of choice.