The pharmacokinetics and metabolism of [3H] cytosine arabinoside in the central nervous system were investigated. In vitro, the ability of rabbit brain slices and the isolated choroid plexus to accumulate [3H] cytosine arabinoside from artificial cerebrospinal fluid (CSF) containing 5 nM [3H] cytosine arabinoside was studied. Although in both tissues the uptake of [3H] cytosine arabinoside was saturable, neither brain slices nor the isolated choroid plexus achieved tissue/medium ratios greater than 1.0 in 30 min. After 15-min incubations iin artificial CSF containing 5 nM [3H] cytosine arabinoside, 8 +/- 2 (S.E.M.;N = 3)% of the 3H within the choroid plexuses was [3H] cytosine arabinoside phosphates; after comparable 30-min incubations, 4 +/- 1 (N = 4)% of the 3H within brain slices was [3H] cytosine arabinoside phosphates. In vivo, 2 hr after the i.c.v. injection into rabbits of [3H] cytosine arabinoside and 0, 2 ro 20 mumol of unlabeled cytosine arabinoside, it was shown that: 1) high concentrations of cytosine arabinoside in the CSF decreased the clearance of cytosine arabinoside from the CSF; 2) cytosine arabinoside was taken up and phosphorylated by brain cells in all regions tested; and 3) although the phosphorylation of [3H] cytosine arabinoside in brain cells was saturable, a portion of the [3H] cytosine arabinoside in brain was phosphorylated to [3H] cytosine arabinoside triphosphate even when the i.c.v. injectate contained 20 mumol of unlabeled cytosine arabinoside.