Binding studies were conducted with intact SH-SY5Y cells, using the mu-opioid-selective antagonist [3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. Previous studies had demonstrated equilibrium binding constants generally higher than those found when binding to membrane preparations for most opioid agonists. Studies on various other G protein-coupled receptors had shown that receptor internalization led to low apparent affinities for agonists and that short or low-temperature incubations could reveal high-affinity binding components. In SH-SY5Y cells, short (1-min) and 4 degrees C incubations did not reveal high-affinity binding components. Nonequilibrium binding experiments could also be used to compare binding affinities of opiate agonists with activity at mu receptors in the cells. SH-SY5Y cells have functional mu and delta receptors. mu Receptors can be measured independently of delta receptors if activity is determined in the presence of the delta antagonist ICI 174,864. When both binding and activity, as measured by inhibition of forskolin-stimulated cAMP accumulation, were conducted for 10 min, the ratio of binding IC50 to activity IC50 could give an indication of the relative efficacy of opioid agonists. These studies indicated that morphine has slightly lower efficacy than etorphine and the peptides DAMGO and DADLE. Each of these compounds has significantly higher efficacy than ethylketocyclazocine and the partial agonist buprenorphine.