Electrotonic coupling of cardiac myocytes at gap junctions may influence patterns of conduction in myocardium. To delineate the three-dimensional structure and distribution of intercellular junctions, we analyzed serial ultrathin sections of canine myocardium with transmission electron microscopy and disaggregated myocytes with scanning electron microscopy. Morphometric analysis of left ventricular myocardium sectioned in three orthogonal planes revealed that 80% of total gap junctional membrane occurred in large, ribbon-like gap junctions oriented transversely at cell end processes. The remaining 20% of gap junctional membrane was contained in small gap junctions located within plicate segments (interdigitating regions of cell-to-cell adhesion) of intercalated disks. In serial ultrathin sections, all gap junctions were contiguous with plicate segments. Thus, true "lateral" gap junctions do not exist in working ventricular myocytes and would not likely be able to withstand shear forces created by laterally sliding cells. Examination of serial plastic sections with light microscopy revealed complex overlapping of myocytes such that individual myocytes were connected at intercalated disks to an average of 9.1 +/- 2.2 other myocytes. These observations provide an improved understanding of the extent and distribution of cell junctions and should facilitate experimental and model studies of conduction in myocardium.