Brain delivery of systemically administered neuropeptide drugs through the tight endothelial cells of brain microvessels, which constitute the blood-brain barrier (BBB), may be achieved by the synthesis of chimeric peptides [Bickel U et al (1993) Proc Natl Acad Sci USA 90:2618]. The peptide drug can be linked by avidin-biotin technology to transport vectors targeting brain, such as the anti transferrin receptor monoclonal antibody, OX26. In the present study we investigated the effects of the coupling procedure on the bioactivity of the μ-opioid receptor specific dermorphin analog Lys 7 -dermorphin (K7DA). It was monobiotinylated at the -amino group of Lys 7 with either a cleavable, i.e. disulfide containing biotinylating reagent, NHS-SS-biotin (sulfosuccinimidyl2-(biotinamido)ethyl-1,3 dithiopropionate), or a noncleavable analog. Biotin-XX-NHS, where XX = bis-aminohexanoyl spacer arm and NHS = N-hydroxysuccinimide. Disulfide cleavage of the biotinylated derivative yields the desbiotinylated peptide, which is thiolated. The biotinylated peptides were coupled to a thiol-ether conjugate of OX26 and either neutral avidin (NLA) or streptavidin. Pharmacologic activity of the biotinylated peptides coupled to the NLA-OX26 vector was investigated with both in vitro μ-opioid radioreceptor assays and in vivo tail-flick analgesia testing following intracerebroventricular injection. The cleavability of the disulfide linker in vivo in rat plasma and brain was assessed with gel filtration HPLC after internal carotid artery perfusion of labeled opioid chimeric peptides. The obtained results are consistent with the following conclusions: (i) opioid peptides have minimal pharmacologic activity when bound to the transport vector, indicating the need for cleavable linkers; (ii) the disulfide linker is stable in vivo in plasma as well as brain capillary endothelial cells, but is rapidly cleaved in rat brain in vivo, indicating that disulfide cleavage occurs beyond the BBB; and (iii) the thiolated peptide released following disulfide cleavage is pharmacologically active at the μ-opioid receptor via a naloxone reversible mechanism, indicating covalent receptor binding is not likely occurring.