Intercellular junctions, identified in freeze-fracture by narrowing of the intercellular gap and codistribution of P-face membrane particles, proliferate during incubation of excised rat prostate tissue in 30% glycerol solution. These junctions fulfill the criteria used to identify gap junctions in freeze-fracture replicas with respect to the size and uniformity of their component particles, the codistribution of the particle aggregates on P-faces of adjacent membranes, and the narrowing of the extracellular cleft at the junction. These gap-junction-like structures form on the lateral surfaces of epithelial cells, where they are normally scarce, within minutes after exposure to glycerol. Glycerol-induced junction formation is not blocked by DNP, a metabolic uncoupler, or by cycloheximide, a protein synthesis inhibitor. Newly formed junctions occur initially in clusters and the number per cluster decreases as individual junctions become larger with longer periods of incubation, suggesting that the clusters coalesce. The structural changes that precede and accompany the formation of these junctions at early times of incubation are comparable to the changes reported to precede the formation of gap junctions accompanying hormonal treatment, development, and other means of natural induction.