Biphalin [(Tyr-D-Ala-Gly-Phe-NH)2] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [125I-Tyr1]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [125I-Tyr1]biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of delta- and mu-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [125I-Tyr1] biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [125I-Tyr1]biphalin was decreased with naloxone, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to delta- and mu-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [125I-Tyr1]biphalin, which could be described by Michaelis-Menten kinetics with a Km of 2.6 +/- 4.8 microM, Vmax of 14.6 +/- 2.89 pmol(-1) x min(-1) x g(-1), and Kd of 0.568 +/- 0.157 microl x min(-1) x g(-1). Brain entry of [125I-Tyr1]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and L-phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.