We utilized the [(35)S]-GTP-gamma-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 + nor-BNI to measure mu receptors, CTAP + nor-BNI to measure gamma receptors and TIPP + CTAP to measure kappa receptors. The assay was first validated with opioid agonists of known subtype specificity (DAMGO for mu, SNC80 for delta, and U69, 593 for kappa receptors). Methadone-stimulated [(35)S]-GTP-gamma-S binding was mu-specific and less potent and efficacious than etorphine (K(d) = 1,537 nM vs. K(d) = 7.8 nM). Buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding but inhibited agonist-stimulated [(35)S]-GTP-gamma-S binding. The antagonist-K(i) values (nM) of buprenorphine at mu, delta, and kappa receptors were 0.088 nM, 1.15 nM, and 0.072 nM, respectively. The antagonist-K(i) values (nM) of naloxone at mu, delta, and kappa receptors were 1.39 nM, 25.0 nM, and 11.4 nM, respectively. Autoradiographic studies showed that buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding in caudate-level rat brain sections but blocked DAMGO-stimulated [(35)S]-GTP-gamma-S binding. In cells expressing the cloned rat mu receptor, buprenorphine was a partial agonist and potent mu antagonist. Administration of buprenorphine to rats produced a long-lasting (>24 h) decrease in mu and kappa2 receptor binding and attenuated mu-stimulated [(35)S]-GTP-gamma-S binding. Viewed collectively, these data indicate that, in this assay system, buprenorphine is a potent mu and gamma receptor antagonist. The clinical implications remain to be elucidated. Synapse 34:83-94, 1999. Published 1999 Wiley-Liss, Inc.