BW373U86 is a potent and highly selective nonpeptidic agonist for delta-opioid receptors. To determine its ability to couple with G protein-linked second messenger systems, this study examined the effects of BW373U86 on the inhibition of adenylyl cyclase and the stimulation of low-Km GTPase activity. In rat striatal membranes, BW373U86 inhibited basal adenylyl cyclase activity in a GTP-dependent manner, with maximal inhibition levels similar to those of the prototypic delta agonist [D-Ser2,Thr6]Leu-enkephalin (DSLET). However, BW373U86 was approximately 100 times more potent than DSLET in inhibiting adenylyl cyclase. Analysis of the inhibitory activity across 10 brain regions revealed that both low and high concentrations of BW373U86 inhibited adenylyl cyclase activity in a manner similar to that of DSLET. Inhibition of adenylyl cyclase by BW373U86 was delta receptor selective, because the delta receptor-selective antagonist naltrindole was significantly more potent than naloxone and the mu receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was ineffective in blocking BW373U86 inhibition. BW373U86 also inhibited adenylyl cyclase activity in membranes prepared from NG108-15 cells, with an IC50 value 5 times lower than that of DSLET. This increased potency was not observed in concentration-effect curves for agonist-stimulated low-Km GTPase in NG108-15 membranes. BW373U86 is a competitive inhibitor of [3H]diprenorphine at delta receptors of NG108-15 cell membranes. However, unlike DSLET, BW373U86 displacement of [3H]diprenorphine binding to NG108-15 cell membranes was not affected by sodium and guanine nucleotides. This lack of GTP effect on binding apparently produced slow dissociation rates for this agonist, because naltrindole was less potent in blocking BW373U86 inhibition of adenylyl cyclase when membranes were preincubated with this agonist. These results demonstrate the novel finding that the binding of a full agonist to a G protein-coupled receptor is not regulated by GTP, and they also show how the lack of regulation in receptor binding affects agonist potency.