When transformed cells were co-cultured with various densities of non-transformed BALB/c 3T3 A31-1-1 cells, the number of transformed cell foci decreased as the density of the A31-1-1 cells was increased. Under the condition of separate co-cultivation in which transformed cloned cells could not make contact with A31-1-1 cells, no inhibitory effect was induced. We examined with a dye-transfer assay the formation of heterologous gap-junctional intercellular communication (GJIC), links between the transformed cells and A31-1-1 cells before and after focus formation. Heterologous GJIC was observed almost always before, but almost never after, focus formation. Using time-lapse photography to record the fate of transformed cloned cells that did not form foci in the co-cultivation, it was noted that most of them were living, but did not proliferate. These results suggested that focus formation of transformed cloned cells was inhibited by contact with non-transformed A31-1-1 cells.