The mechanisms implicated in the vectorial flux of nucleosides across polarized epithelia have not yet been characterized appropriately, neither is it known how hCNT3, a nucleoside transporter expressed in several segments of the nephron [1], contributes to this process. To address this issue, we have first used and characterized a cell line derived from murine proximal convoluted tubule (PCT), which expresses CNT3 as the only murine ortolog of the SLC28 gene family. PCT cells, when grown on transwell filters, show CNT3-type transport activity at the apical side. In the presence of sodium, H3-cytidine apical to basal flux is quantitatively relevant (240 ± 12 pmol/mg prot min) and associates with considerable nucleoside metabolism, thus resulting in uracil and uridine as major metabolites detected at the basal compartment. In the absence of sodium (CNT3 being inactive) a significant reduction in vectorial flux was found (60 ± 2 pmol/mg prot min, P>0.001 vs sodium), although most of the tracer at the basal side turned out to be the intact substrate, H3-cytidine. These data were mimicked in another cell model, MDCK cells, transiently transfected with the human ortolog of CNT3 (hCNT3). When using a GFP-tagged hCNT3 expression, and its corresponding functional activity, was found to be exclusively located at the apical side of the monolayer [2], in accordance with what we have also found for the endogenously expressed CNT3 of murine PCT cells. Apical insertion of hCNT3 results in a vectorial flux and nucleoside metabolism similar to that found in PCT cells in the presence of sodium, whereas data in the absence of the cation resembled those found in MDCK cells transfected with the empty vector. This cell model was further used to determine how hCNT3 insertion contributed to the vectorial flux of a panel of antiviral and antineoplasic nucleoside-derived drugs. Our data demonstrate that apical insertion of hCNT3 and, therefore, hCNT3 function itself, are major determinants of nucleoside and nucleoside-derived vectorial flux across epithelia.