The formation of tight junctions can be induced in the human adenocarcinoma cell line HT 29 by treatment with trypsin at 37°C. In contrast, after treatment of the cells with trypsin at low temperature (3°C), no tight junctions were observed. However, abundant formation of tight junctions occurred when cells were treated with trypsin at 3°C, washed with soybean trypsin inhibitor, and subsequently incubated at 37°C. Thus, this protocoi allows for the first time the temporal separation of the induction and assembly of tight junctions.