A number of models have been proposed to explain the organization of intramembrane fibrils at tight junctions. These include (1) the two-fibril model of Chalcroft and Bullivant, (2) a later revision of this model by Bullivant (the offset two-fibril model), (3) Staehelin's model (similar to the two-fibril model, but it implies a different interpretation of the fracture faces), and (4) the single-fibril model of Wade and Karnovsky. Studies of these models using conventional freeze-fracture and the complementary replica methods have not been conclusive. In an attempt to determine which model holds in vivo, we have utilized the quick-freeze, freeze-fracture method. Tight junctions between hepatocytes were split by perfusion with hypertonic saline, or by incubation in Ca-free EGTA saline. The tissue was then fixed, quick frozen, freeze fractured, deep etched, and rotary shadowed. Thus both the true surface and fracture faces of tight junctions could be viewed stereoscopically by tilting the replicas. Control specimens were simply fixed and prepared as above for viewing without splitting of the tight junctions. On the P faces of split tight junction preparations the strands were discontinuous and particulated. They clearly retained the height of the control P face strands, however, and they were as high or slightly higher than the true surface gentle ridges with which they seemed to correspond. No lines of prominent bumps or defects were observed on top of the true surface ridges. In a second approach fresh liver was quick frozen without fixation or cryoprotection; these samples were then freeze fractured and unidirectionally shadowed. Stereoscopic examination of the replicas revealed a network of broad linear elevations and intervening valleys on the P face, with shallow grooves running along the top of the linear elevations. Occasionally these grooves were interrupted by elevated particles left behind during fracture, and the grooves sometimes even became inverted into P face strands, especially near a transition of the fracture plane from P face to E face. Examination of the E face of unfixed specimens revealed a network of particulated strands. The occasional particles and strands of the P face were clearly lower than the strands on the E face. These data support the offset two-fibril model of Bullivant.