Previous studies have demonstrated that peptidic and nonpeptidic delta-opioid receptor agonists have different effects depending on the measure. For example, nonpeptidic delta-opioid agonists, but not peptidic agonists, produce convulsions in rats, and in vitro studies suggested that peptidic and nonpeptidic delta-opioid agonists might have differential mechanisms of receptor downregulation. The present study evaluated potential differences between peptidic and nonpeptidic delta-opioid agonists in their ability to activate G proteins using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) autoradiography experiments in rat brain slices. The peptidic agonist [d-Pen(2),d-Pen(5)]-enkephalin and the nonpeptidic agonist (+)BW373U86 [(+)-4-[alpha(R)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide] demonstrated concentration-dependent increases in [(35)S]GTPgammaS binding that were attenuated by the delta-opioid antagonist naltrindole. (+)BW373U86 was more potent and efficacious than the peptidic agonist, and this difference remained consistent across brain regions where significant stimulation was observed. In addition, multiple delta-opioid compounds were evaluated for their agonist activity in this assay. These data suggested that differences between peptidic and nonpeptidic delta-opioid agonists in behavioral studies were most likely caused by differences in agonist efficacy. Finally, these data also revealed that [(35)S]GTPgammaS autoradiography could be used to compare efficacy differences among agonists across various brain regions in rat brain slices.