Peptide hormones and neurotransmitters play crucial roles in the maintenance of physiological function at both the cellular and organ level. Although peptide neuropharmaceuticals have enormous potential in the treatment of disease states, the blood-brain barrier (BBB) generally prevents the entry of peptides into the brain either by enzyme degradation or by specific properties of the BBB. Peptides that act at opioid receptors are currently being designed for analgesia and to reduce the unwanted side effects associated with morphine, such as addiction and inhibition of gastric motility. It has been the focus of our group to produce stabile peptide analogues of Met-enkephalin, that lead to analgesia without side effects. In this paper we present the methodologies that have been used to elucidate the transport mechanisms of three peptides across the BBB. Using a primary endothelial cell culture model of the BBB, in situ perfusion, and kinetic analysis we show that D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) crosses the BBB via diffusion, [D-penicillamine2,5]enkephalin uses a combination of diffusion and a saturable transport mechanism, and biphalin ([Tyr-D-Ala-Gly-Phe-NH]2) uses diffusion and the large neutral amino acid carrier. Understanding BBB transport mechanisms for peptides will aid in the rational design of peptides targeted to the brain.