By comparing in situ negative staining of mammalian heart muscle using La(NO3)3with conventional positive staining by uranium and lead salts, we showed that 1) the membrane area of rat cardiac gap junctions (GJs) at the intercalated disks is threefold to fourfold greater than previously thought; 2) connexon arrays of cardiac GJ are subdivided into microdomains by connexon-free aisles; 3) profiles of GJ-associated vesicles (GJAVs) of plasmalemmal origin (which are present extracellularly and sharply localized at three extracellular sites) are paired to form GJs with each other and with myocyte plasmalemma; 4) some GJAVs contain arrays of assembled connexons; and 5) myocytes contain intracytoplasmic complexes lying within cylindrical or cigar-shaped membranes and consisting of GJs and multiple vesicles apparently dissociating from these GJs.