The permeability of the blood-cerebrospinal fluid (CSF) barrier to ³H-labelled thyrotropin-releasing hormone (TRH), was studied at the blood-tissue interface of the isolated perfused choroid plexus of the sheep, using a rapid (if< 30 s), single circulation paired-tracer dilution technique, in which d-[¹⁴C]mannitol serves as an extracellular marker. Arterio-venous loss of¹⁴C radioactivity reflects the percentage of the d-mannitol dose that crosses the blood-CSF barrier using a non-specific pathway. This loss suggests that the choroidal epithelium is moderately leaky. Cellular uptake of TRH, estimated by directly comparing venous dilution profiles of [³H]TRH and d-[¹⁴C]mannitol was independent of this leakiness. The unidirectional transport of TRH could not be saturated with unlabelled TRH at a concentration as high as 10 mM, but was markedly reduced by 10 mM proline and by inhibitor of amidase and aminopeptidase activity, bacitracin (2 mM). Permeability of the blood-brain barrier to [³H]TRH was studied in the adult rat, employing the intracarotid injection technique of Oldendorf ²⁵ in which [¹⁴C]butanol served as an ‘internal standard’. Brain-uptake of ³H radioactivity corrected for residual vascular space indicated a low extraction from the blood of TRH during a 15 s period of exposure to the peptide. Self-inhibition of [³H]TRH uptake by unlabelled TRH (10 mM) could not be demonstrated, but l-proline (10 mM) and bacitracin (2 mM) strongly inhibited this uptake. It is concluded that similar mechanisms at the blood-CSF and blood-brain barriers tend to prevent TRH penetration into the brain; namely the absence of significant transport systems or binding at the barrier and peptide degradation by the barrier or by plasma enzymes. However, in the presence of bacitracin, which prevents deamidation and hydrolysis of TRH, there is still an arterio-venous loss of ³H radioactivity during a single capillary passage through the choroid plexus of the same order as that of d-[¹⁴C]mannitol. It is suggested that there may be a passage of some intact TRH by a non-specific pathway through the choroid plexus into the CSF. This passage from blood via the CSF followed by diffusion in the extracellular space of nervous structures adjacent to the ventricles may represent a route of entry for TRH into the brain.