Tumor-promoting phorbol esters reversibly inhibit intercellular communication between BALB/c 3T3 cells. In order to study the possible role of blocked intercellular communication in the promotion step in cell transformation, we investigated the effect of phorbol ester tumor promoters on cell transformation and intercellular communication in BALB/c 3T3 A31-1-1 cells by a dye-transfer method. When the cells are in the growing phase, inhibition of dye transfer by phorbol esters is complete but transient; more than 90% inhibition was observed 4 h after treatment of the cells with either 12-O-tetradecanoylphorbol-13-acetate or phorbol-12,13-didecanoate, but the extent of dye transfer returned to the control level after 24 h of treatment. However, when these phorbol ester-treated cells were cultured beyond confluence in the presence of tumor promoters, the capacity to transfer dye decreased again and was inhibited continuously for at least 5 weeks of culture. In control cultures, the extent of dye transfer between cells did not decrease at their confluence. The ability of 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-didecanoate to induce continuous inhibition of dye transfer between these cells correlated well with their capacity to promote transformation of BALB/c 3T3 cells initiated with 20-methylcholanthrene. These results suggest that the continuously blocked intercellular communication after confluence, rather than its transient inhibition during the growing phase, might play an important role in the promotion of in-vitro two-stage transformation of BALB/c 3T3 cells.