A method is reported for isolating a preparation of hepatic gap junctions from the mouse. The method involves a collagenase digestion, treatment with the detergent Sarkosyl NL-97, and ultrasonication, followed by sucrose gradient ultracentrifugation. A run with 36 animals yields 0.1-0.5 mg protein. Electron microscopy with thin-sectioning and negative staining techniques reveals that the final pellet is a very pure preparation of gap junctions, accompanied by a small amount of amorphous contamination. Polyacrylamide-gel electrophoresis of sodium dodecyl sulfate (SDS)-solubilized material shows one major protein in the junction, with an apparent mol wt of 20,000, and two minor components. Thin-layer chromatography demonstrates one major and one minor phospholipid, and some neutral lipid. Low-angle X-ray diffraction of wet and dried specimens show reflections which index on an 86 A center-to-center hexagonal lattice, corresponding closely to electron microscope data. Dried specimens also show a lamellar diffraction, corresponding to the total profile thickness of the junction (150 A).