The permeability of the blood-brain barrier to [ tyrosyl-3,5-³H]enkephalin-(5-l-leucine) (abbreviated to Leu-Enk) and of its synthetic analogued-alanine²-[ tyrosyl-3,5-³H]enkephalin-(5-d-leucine) (abbreviated tod-Ala²-d-Leu⁵-Enk) was studied, in the adult rat, by means of Oldendorf's²⁷ intracarotid injection technique. The brain uptake index (BUI) corrected for residual vascular radioactivity was about the same for both peptides, indicating a low extraction from the blood during a 5- or 15-s period of exposure to the peptides. Transport of Leu-Enk was not saturated by unlabelled Enk at a concentration as high as 5 mM but was completely abolished by 5 mM tyrosine and by the inhibitor of aminopeptidase activity, bacitracin (2 mM). Also the typicall-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2 car☐ylic acid (BCH)⁹ at 10 mM concentration markedly reduced (by 80%) Leu-Enk uptake by the brain. In contrast, brain uptake ofd-Ala²-d-Leu⁵-Enk was reduced only to about one-half of its control value by bacitracin or by 25% by BCH. Brain uptake forl-tyrosine was typically large and markedly inhibited by BCH but not inhibited by 5 mM unlabelled Leu-Enk. These results show that the measurable but low first-pass extractions for enkephalins are not representative of the uptake of these peptides into the brain, but rather reflect their extreme sensitivity to enzymatic degradation with a release of the N-terminal tyrosine residue. The results also suggest that small amounts ofd-Ala²-d-Leu⁵-Enk might cross the blood-brain barrier in an intact form. It is concluded that the absence of a transport mechanism at the blood-brain barrier, together with a rapid enzymatic degradation, tends to prevent significant penetration of the barrier by Leu-Enk during the 5- or 15-s period of these studies.d-Ala²-d-Leu⁵-Enk however, shows a penetrance of the blood-brain barrier significantly greater than that of sucrose.