The localization and mechanism of thymidine and deoxyuridine transport in the central nervous system were studied in vivo and in vitro. Previous studies have shown that thymidine enters brain from blood in part via the CSF. In vitro, isolated adult bovine cerebral microvessels, which readily concentrated and phosphorylated deoxyglucose, were unable to concentrate thymidine and deoxyuridine. In vivo, [3H]thymidine (0.2 microM) and [3H]deoxyuridine (0.4 microM) were not extracted more readily than [14C]sucrose in a single pass through the cerebral circulation of rats. In vivo, [3H]thymidine retention in CSF and brain after entry from blood was increased when the efflux of [3H]thymidine from CSF and the phosphorylation of [3H]thymidine in brain were depressed by the intraventricular injection of unlabeled thymidine. These studies and previous work suggest that the transfer of thymidine (and deoxyuridine) through the blood-brain barrier in either direction must be extremely low. The present studies are consistent with the postulate that thymidine is transported by an active transport system in the choroid plexus that transfers thymidine from blood into the CSF; from the CSF, the thymidine enters brain cells and is phosphorylated.