Adenovirus (Ad) vectors are widely used in gene therapy and in vitro/in vivo gene transfer. However, Ad-mediated gene transfer in epithelial cells shows low efficiency, because Ad fiber cannot bind to the primary receptor, the coxsackievirus and adenovirus receptor (CAR), present in tight junctions. Caco-2 monolayer cells cultured on Transwell-chamber plates for approximately 2 weeks are widely used for drug membrane permeation studies, but Ad-mediated gene transfer is difficult in Caco-2 monolayer cells. First, we examined the efficiency of gene transfer into Caco-2 monolayer cells. Luciferase production in cultured Caco-2 cells transduced with Ad vectors was 20-fold lower on day 12 than on day 1. In contrast, the expression of CAR protein in Caco-2 cells gradually increased along with the duration of culture. For efficient gene transfer into Caco-2 monolayer cells, the binding ability of Ad vectors with CAR was found to be important. Capric acid (C10), a medium-chain fatty acid is a tight-junction modulator used as a pharmaceutical agent. We found that a novel gene transfer method using transduction with Ad vectors in the presence of C10 led more efficiently to LacZ expression in Caco-2 monolayer cells than Ad vectors alone. The results of the present study indicate that C10 could be very useful for Ad-mediated gene transfer in human colonic Caco-2 epithelial cells.