The mechanisms by which biotin enters and leaves brain, choroid plexus and cerebrospinal fluid (CSF) were investigated by injecting [3H]biotin either intravenously or intraventricularly into adult rabbits. [3H] biotin, either alone or together with unlabeled biotin was infused at a constant rate into conscious rabbits. At 180 minutes, [3H]biotin had entered CSF, choroid plexus, and brain. In brain, CSF, and plasma, greater than 90% of the nonvolatile 3H was associated with [3H]biotin. The addition of 400 mumol/kg unlabeled biotin to the infusion syringe decreased the penetration of [3H]biotin into brain and CSF by approximately 70 percent. Two hours after an intraventricular injection, [3H]biotin was cleared from the CSF more rapidly than mannitol and minimal metabolism of the [3H]biotin had occurred in brain. However, 18 hours after an intraventricular injection, approximately 35% of the [3H]biotin remaining in brain had been covalently incorporated into proteins, presumably into carboxylase apoenzymes. These results show that biotin enters CSF and brain by saturable transport systems that do not depend on metabolism of the biotin. However, [3H]biotin is very slowly incorporated covalently into proteins in brain in vivo.