Binding of cis-(+)-3-methylfentanyl isothiocyanate (SUPERFIT) to cloned opioid receptors stably expressed in Chinese hamster ovary cells was characterized. SUPERFIT inhibited [3H]diprenorphine binding with much higher affinity for the δ than the μ or κ receptor. Pretreatment with SUPERFIT followed by extensive washing reduced 8 binding with an IC50 value of 7.1 nM, yet it did not affect μ and κ binding up to 0.l μM. The reduction in g binding by SUPERFIT pretreatment was due to a decrease in B(max) with no change in K(d). These results indicate that SUPERFIT is a highly selective 8 irreversible ligand. We then determined the region in the δ receptor that confered binding selectivity for SUPERFIT by examining its binding to six μ/δ chimeric receptors. SUPERFIT bound to δ, μ/δ1 (amino acids μ1- 94/δ76-372), δ/μ3 (δ1-134/μ154-398), and δ/μ4 (δ1-187/μ207-398) receptors with high affinity but to μ, δ/μ1 (δ1-75/μ95-398), μ/δ3 (μ1-153/δ135-372), and μ/δ4 (μ1-206/δ188-372) receptors with low affinity. Pretreatment with SUPERFIT potently inhibited [3H]diprenorphine binding to δ, μ/μ1, δ/μ3, and δ/μ4 but affected binding to μ, δ/μ1, μ/δ3, and μ/δ4 only at much higher concentrations. Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane helix of the δ receptor is important for selective binding of SUPERFIT.