We utilized the [ 35 S]-GTP-␥-S functional binding assay to determine the selectivity of opioid receptor agonists in guinea pig caudate membranes. The study focused on two opioid agonists used for treating opioid-dependent patients: methadone and buprenorphine. Selective antagonists were used to generate agonist-selective conditions: TIPP ϩ nor-BNI to measure µ receptors, CTAP ϩ nor-BNI to measure ␥ receptors and TIPP ϩ CTAP to measure receptors. The assay was first validated with opioid agonists of known subtype specificity (DAMGO for µ, SNC80 for ␦, and U69,593 for receptors). Methadone-stimulated [ 35 S]-GTP-␥-S binding was µ-specific and less potent and efficacious than etorphine (K d ϭ 1,537 nM vs. K d ϭ 7.8 nM). Buprenorphine failed to stimulate [ 35 S]-GTP-␥-S binding but inhibited agonist-stimulated [ 35 S]-GTP-␥-S binding. The antagonist-K i values (nM) of buprenorphine at µ, ␦, and receptors were 0.088 nM, 1.15 nM, and 0.072 nM, respectively. The antagonist-K i values (nM) of naloxone at µ, ␦, and receptors were 1.39 nM, 25.0 nM, and 11.4 nM, respectively. Autoradiographic studies showed that buprenorphine failed to stimulate [ 35 S]-GTP-␥-S binding in caudatelevel rat brain sections but blocked DAMGO-stimulated [ 35 S]-GTP-␥-S binding. In cells expressing the cloned rat µ receptor, buprenorphine was a partial agonist and potent µ antagonist. Administration of buprenorphine to rats produced a long-lasting (Ͼ24 h) decrease in µ and 2 receptor binding and attenuated µ-stimulated [ 35 S]-GTP-␥-S binding. Viewed collectively, these data indicate that, in this assay system, buprenorphine is a potent µ and ␥ receptor antagonist. The clinical implications remain to be elucidated.