We have combined freeze-fracture and electrophysiological methods in a study of gap junction formation between reaggregated Novikoff hepatoma cells. Cell clumps are dissociated with EDTA, and the resulting single cells are allowed to reaggregate (5-180 min) in loose pellets in the presence of calcium at 37°. The earliest electron microscopic evidence for the genesis of new junctions is the appearance of flattened regions of the plasma membrane with a relative paucity of small intramembranous particles. These regions contain instead loosely organized groupings of 9- to 11-nm intramembranous particles, which are seen on the A face of the fractured plasma membrane, while corresponding pits occur on the membrane B face. We have termed the specialized membrane regions "formation plaques." They are seen as early as 5 min after reaggregation and are quite numerous by 30 min. Larger plaques are observed at later times. Plaques seen at 30 min are consistently matched with other plaques on apposed cells, although the extracellular space separating these structures still exceeds 10 nm. By 60 min, some matched plaques display a reduced extracellular space, resembling that of normal gap junctions. Between 30 and 60 min, aggregates of closely packed particles on A faces and hexagonally arranged pits on B faces frequently appear in the formation plaques. The aggregates, which are indistinguishable from small gap junctions, appear to enlarge over the subsequent 2-hr period as the number of unaggregated 9- to 11-nm particles declines. Microelectrode studies demonstrate progressive increases in the percent of interfaces containing lowresistance junctions and in the degree of elctrical coupling in preparations incubated up to 2 hr. Coupling is first detected at about the same time as particle aggregates (or formation plaques with reduced extracellular spaces), and increases as aggregate sizes increase.