After application of [3H]benzylpenicillin ([3H]BP) in lateral brain ventricle in dogs, the distribution of [3H]BP to contralateral ventricle and cisterna magna was much higher when its active transport from cerebrospinal fluid (CSF) was blocked by probenecid than under control conditions. Analysis of [3H]BP concentrations in both lateral ventricles and cisterna magna over time indicates that active transport restricts distribution of substances along CSF spaces and contributes to the maintenance of their concentration gradients between CSF compartments. This suggests that biochemical changes in a part of the brain and the adjacent CSF compartment may not be reflected into remote compartments of CSF such as lumbar CSF if substances in question are removed from CSF by active transport.