In polarized epithelia, the tight junctions (TJs) constitute a barrier that controls the paracellular flux of solutes and water. In the renal LLC-PK1 cells, the TJ permeability can be correlated directly with the unidirectional transepithelial flux of solutes, such as D-mannitol, which have negligible affinity for cell membrane transport systems, and inversely to the transepithelial electrical resistance (TER). This study investigates TJ permeability and cell proliferation in LLC-PK1 cells treated with the phorbol ester, tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) or with epidermal growth factor (EGF), a mitogen without secondary carcinogenic effects. Both TPA and EGF induced mitogenesis in LLC-PK1 cells. The TJs in TPA-treated cells were leaky, as indicated by decreased TER, increased D-mannitol flux, and TJ penetration by ruthenium red. In contrast, EGF treatment did not result in a decrease in TER, only slightly increased the D-mannitol flux, and did not result in ruthenium red penetration of the TJs. This inability of ruthenium red to penetrate TJs between EGF-treated epithelial cells was true even for cells in mitosis. The data therefore indicate that mitogenesis per se does not increase TJ permeability, suggesting that the TJ leakiness observed during tumor promotion with phorbol esters does not arise from cell proliferation and is perhaps associated distinctly with or causal to the transformation of those cells.