In order to quantitate the extent to which opioid agonist potencies obtained in behavioral assays are determined by the apparent in vivo affinity and efficacy of the agonist, the antinociceptive effects of the mu opioid agonists morphine, fentanyl, etonitazene, and NIH 10741 were assessed before and after administration of the insurmountable mu opioid antagonist clocinnamox (CCAM) in a 55 degrees C warm-water tail withdrawal test in Swiss albino mice. Under control conditions, all four mu opioid agonists produced a full antinociceptive response with the following ED50 values (in mg/kg): morphine, 12; fentanyl, 0.47; etonitazene, 0.039; NIH 10741, 0.0051. Analysis of CCAM's effects according to Black and Leff gave the following agonist efficacy or tau values: Morphine, 4; fentanyl 15, etonitazene, 7; and NIH 10741, 59. The respective KA values were (in mg/kg): morphine, 29; fentanyl, 7.3; etonitazene, 0.22; and NIH 10741, 0.30. The major determinant of the experimentally observed ED50 values seemed to be the apparent in vivo affinity of the respective agonist and not its efficacy. KA values (expressed as mol/kg) correlated with the Ki values (in mol/l) obtained with [3H]DAMGO radioligand binding (r = 0.96 for pKA vs. pKi), although being on average 11,000-fold higher. Values for q, the available receptor fraction as determined in the behavioral experiments, correlated strongly (r = 0.96) with the q values determined by ex vivo [3H]DAMGO- and [3H]naltrexone equilibrium binding (i.e., Bmax,clocinnamox/Bmax,control), the relationship approaching unity.