Oral administration of tetrahydrobiopterin (BH(4)) has been known to be effective in treating BH(4)-deficient patients. It has long been established that BH(4) is absorbed by the intestinal mucosa. However, the mechanism for translocation of BH(4) across epithelial cells has not been elucidated. In order to study BH(4) transport mechanisms, Caco-2 cells were employed in this study as an epithelial cell model. Caco-2 cells were cultured (2 x 10(4) cells/0.3 cm(2) well) for 21 days in a 24-well format using Transwell, a porous membrane-based culture dish, at which point they had established themselves as a tight sheet with a definite polarity. When BH(4) (100 micromol/L) was given to cells from the apical side, a considerable translocation toward their basolateral side was noted. The rate of BH(4) movement was around 150 pmol/h per well. This was comparable to the highest rate of BH(4) uptake or its release so far obtained using a monolayer culture of Caco-2 cells on an ordinary plastic plate. The transcellular movement of BH(4) across the polar culture on the porous membrane was effectively prevented by benzbromarone (10 micromol/L), a well known inhibitor of a group of transporters including urate transporter (URAT1), organic anion transporters (OATs), and multidrug-resistance-associated proteins (MRPs). It was thus concluded that in Caco-2 cells, BH(4) moved across the cell interior in a rapid ligand-specific manner that was driven by a transporter.