The aim of this study was to investigate the expression of nucleoside transporters in rat choroids plexus epithelial cells (RCPEC) in primary culture from humanely killed rat. Classical PCR analysis revealed that RCPECs contain mRNA for equlibrative transporters (rENT) 1 and 2 and concentrative transporters (rCNT) 2 and 3. This result was refined using real time PCR analysis which showed that relative expression of the rENT1 and rENT2, mRNA to that of beta-micro globulin were lower in RCPECs than in whole brain homogenate, whereas mRNA for rCNT2 and rCNT3 were similar in RCPECs to that in whole brain homogenate. Western blot analysis of RCPEC membranes revealed the presence of ENT1, ENT2 and CNT2 proteins, which were present in both heavily glycosylated and non-glycosylated form. Distribution of these proteins was investigated by measuring nucleoside uptake into RCPECs grown on plastic permeable supports. Cellular uptake of [14C] adenosine from the upper chamber (corresponding to the CSF compartment) was reduced by >60% by replacing Na+ with choline in the medium in that chamber, reduced significantly by 0.01mM formycin B or tubercidin (p<0.01, n=4 and p0.05, n=4). Uptake of [14C] adenosine from the lower chamber (corresponding to the CP interstitial fluid side) was not affected (p>0.05, n=5) by Na+ replacement with choline, but was reduced by approximately 50% by 1×10-6M NBTI (p<0.01, n=4). Addition of 0.05mM hypoxanthine, a nucleobase that does not show affinity for nucleoside transporters except ENT2, caused significant inhibition of [14C] adenosine uptake into RBECs from either chamber (p<0.05). These results suggest that the Na+-independent, equilibrative transporter, ENT1, is present at the cellular membrane corresponding to the CP interstitial fluid side whilst the Na+-dependent, concentrative transporter, CNT2, is present on the opposite membrane, facing CSF in vivo; ENT2 appears to be present on both membranes. These data suggest that the main role of nucleoside transporters at the RCPEC might be the efflux transport of purine nucleosides and nucleobase hypoxanthine from the CSF into the blood.