Tight junctions (TJ) of the fascia occludens type can be rapidly assembled in the human colon adenocarcinoma cell line HT 29 under the influence of trypsin or ammonium sulfate. We have studied the influence of the metabolic inhibitors, dinitrophenol (DNP) and deoxyglucose (DG), on the induced formation of TJ in this cell line. A reduction of the ATP level by DG treatment to 20% of control values did not affect the amount and complexity of induced TJ fibrils. However, under conditions of severe ATP depletion obtained by DNP, the velocity of TJ formation was substantially reduced, and the arrangement of the TJ fibrils as observed by freeze-fracture electron microscopy showed characteristic changes.