Treatment of the kidney epithelial cell line MDCK with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a large increase in the permeability of tight junctions within 2-4 hr. This change in transepithelial permeability is accompanied by alterations in cell morphology that include the opening of tight junctions, loss of apical surface microvilli and the formation of cytoplasmic processes containing numerous microtubules, microfilaments and 10 nm intermediate filaments. Simultaneously, TPA also stimulates a 10-20 fold increase in the synthesis and secretion of the serine protease plasminogen activator (PA); however, the inhibition of PA synthesis by cycloheximide does not prevent the morphological changes or the increased tight junctional permeability, suggesting that PA is not involved in these early TPA-induced events. Drugs that inhibit cytoskeletal function, such as cytochalasins B and D, colchicine, and vinblastine sulfate, are all relatively effective in preventing the TPA-induced morphological changes but not the increases in transepithelial permeability. Our results provide evidence that possible sites of TPA action include epithelial tight junctions as well as components of the cytoskeleton.