Previous studies in the biotin-deficient rat have shown that brain biotin concentrations and the activity of biotin-dependent carboxylases are relatively preserved in the face of biotin starvation and systemic biotin deficiency. These data suggested the existence of a concentration mechanism for biotin in brain, and the present studies were undertaken to further characterize brain biotin transport. We presently show that rat cerebrospinal fluid biotin concentrations are 2.5 times higher than serum concentrations, consistent with the existence of a concentrative mechanism for biotin. Further, we demonstrate uptake of 3H-biotin into rat brain from blood at physiologic biotin concentrations, using single pass clearance measurements of a brain uptake index. The calculated brain uptake indices for biotin, and the inhibition kinetics, are consistent with the possible existence of a low affinity mediated uptake mechanism. The results have implications for the pathophysiology of human biotin-responsive multiple carboxylase deficiency.