In rabbits, the efflux of intraventricularly injected methotrexate from the CSF was retarded by pretreatment with 200 mg/kg of i.p. probenecid. In vitro, the ability of the isolated choroid plexus to concentrate the antitumor agent methotrexate was depressed by the inclusion of probenecid in the incubation medium. These experimental results are consistent with the hypothesis that probenecid depresses the clearance of methotrexate from the CSF by blocking the transport of methotrexate from CSF to blood via the choroid plexus.