The major side effect of morphine and its active metabolite, morphine-6-glucuronide (M6G), is respiratory depression, which is mediated by mu opioid receptors in the medulla and pons. Although the effect of morphine on coupling between mu opioid receptors and G proteins has been studied, the effect of M6G on this coupling has not. Therefore, stimulation of guanylyl-5'-O-([gamma(35)S]-thio)-triphosphate ([(35)S]-GTPgammaS) binding by these two narcotic analgesic drugs was compared to the mu-specific synthetic opioid peptide [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin in Chinese hamster ovarian cells stably transfected with the murine mu opioid receptor and in brainstem membranes prepared from 3-, 7-, and 14-day-old guinea pigs. All three agonists stimulated [(35)S]-GTPgammaS binding in transfected cells and neural tissue, and the stimulation was antagonized by naloxone. In brainstem membranes, but not transfected cells, M6G was less efficacious but more potent than morphine, which may be due to differences between murine and guinea pig mu opioid receptors or in the G proteins in these two tissues. Efficacy of the agonists did not change during development, but overall potency decreased between 3 and 14 days after birth. In vivo potency differences for respiratory depression between morphine and M6G are qualitatively similar to in vitro potency differences of these drugs to stimulate [(35)S]-GTPgammaS binding in neonatal guinea pig brainstem membranes. Tolerance to opioid effects on [(35)S]-GTPgammaS binding developed in transfected cells incubated with morphine with the maximum decrease in potency occurring 18 h later than the maximum decline in efficacy.