An iodine-labelled derivative of β-endorphin, <sup>125</sup>I-Tyr<sup>27</sup> β<sub>h</sub>-endorphin, was used in carrier-free form to study the binding of β-endorphin to brain opioid receptors. The experiments were carried out with rat cortex membranes in vitro under conditions that gave a high degree of naloxone reversible binding. β<sub>h</sub>-Endorphin and nonradioactive iodo-Tyr<sup>27</sup> β<sub>h</sub>-endorphin were found to be identical in their ability to inhibit the binding of <sup>l25</sup>I-Tyr<sup>27</sup> β<sub>h</sub>-endorphin. Competition experiments demonstrated the existence of binding sites with higher affinity for β-endorphin than for a variety of other opioids, including naturally occurring fragments of β-endorphin. The experiments show that <sup>125</sup>I-Tyr<sup>27</sup> β-endorphin possesses similar binding properties to the unmodified peptide and can be used with the advantages of iodine-<sup>125</sup> as an isotope for the investigation of β-endorphin receptors in brain. In experiments employing <sup>125</sup>I-Tyr<sup>27</sup> β-endorphin 1 -27 as the radioiodinated ligand, binding curves were obtained which showed that β-endorphin 1–31 was more potent than β-endorphin 1–27 in inhibiting the binding of the labelled 27 residue peptide. With both the 27 and 31 residue radioligands, magnesium ion enhanced the specific binding whereas sodium ion and guanylyl-imidodiphosphate had a strong inhibitory effect. The data indicate that β-endorphin 1–27 binds with reduced affinity to the same receptors as β-endorphin 1–31 and like the 31 residue peptide exhibits properties characteristic of an agonist.