Connexin32, the major protein that forms gap junction channels in rat liver tissue, was solubilized and purified by immunoaffinity chromatogrnphy. A monoclonal antibody recognizing a cytoplasmic domain of connexin32 was immobilized onto CNBr-activated Sepharose beads. The immunobeads were used for one-step purification of connexin32 from the Triton X-100 solubilized plasma membrane fraction of rat liver tissue. The connexin32 obtained by this immunoaffinity method was highly pure. In contrast to the conventional methods that isolate an intact gap junction membrane, this rapid procedure does not expose the protein to denaturing conditions, and yields connexin32 which can be readily incorporated into liposomes for use in reconstitution studies. Structural studies with gel-filtration chromatography and negative-staining electron microscopy showed that the immunoaffinity-purified connexin32 is present as hexameric forms.