This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+-dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+-independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.