The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.