A bile canaliculus-derived preparation containing junctional complexes has been obtained from mouse livers using subcellular fractionation techniques. The junctional complexes include structurally intact zonulae occludentes (ZOs). Extraction of this preparation with the anionic detergent sodium deoxycholate (DOC) left junctional ribbons, the detergent-insoluble zonular remnants of the junctional complexes. When visualized in negative stain electron microscopy, each of these ribbons contained a branching and anastomosing network of fibrils which appears similar to that of ZOs in freeze-fractured whole liver. Comparative measurements of freeze-fracture and negative stain fibril diameters and network densities support this relationship. SDS polyacrylamide gel analysis shows the DOC-insoluble junctional ribbons to be characterized by major polypeptides at 37,000 and at 48,000, with minor bands at 34,000, 41,000, 71,000, 86,000, 92,000, and 102,000. The ZO-containing membrane fractions have been isolated in the presence of EGTA in concentrations and under conditions shown by others to disrupt normal ZO morphology and physiology in whole living epithelia. The network of fibrils visualized in these fractions by negative staining is structurally resistant to treatment with DOC, but is either solubilized or disrupted by N-lauroylsarcosine.