The highly potent µ-opioid receptor agonist 14-methoxymetopon (4,5α-epoxy-3-hydroxy-14β-methoxy-5β,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the µ-opioid receptor with high potency, while selective κ-opioids and δ-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPγS binding was inhibited by naloxone and selective µ-opioid antagonists, indicating a µ-opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide µ-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing µ-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level.