By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved . Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons . In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays . By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling .These experiments demonstrate that, contrary to the conclusions of some earlier studies