Phenotypic differences in analgesic sensitivity to codeine (3-methoxymorphine) results from polymorphisms in cytochrome P450 -2D6, which catalyzes O-demethylation of codeine to morphine. However, O-demethylation reportedly is not required for analgesic activity of the 7,8-saturated codeine congeners dihydrocodeine, hydrocodone, and oxycodone. This study determined the potency and efficacy of these compounds and their demethylated derivatives to stimulate -and ␦-opioid receptor-mediated G-protein activation using agonist-stimulated guanosine 5Ј-O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding. Results showed that 7,8-saturated codeine congeners were more efficacious than codeine in activating -receptors, but only dihydrocodeine was more efficacious at ␦-receptors. Hydrocodone and oxycodone were ϳ10-fold more potent than codeine and dihydrocodeine at either receptor. Morphine-like compounds with a 3-hydroxy group were ϳ30-to 100-fold more potent than their 3-methoxy analogs at the -receptor, and these compounds generally exhibited greater efficacy (e.g., morphine produced 2-fold greater maximal stimulation than codeine). Removal of the N-methyl group did not affect efficacy or potency of codeine congeners to activate -receptors, whereas this modification generally increased efficacy but decreased potency of morphine congeners. At the ␦ receptor, morphine congeners showed greater potency and structuredependent differences in efficacy compared with codeine congeners, whereas removal of the N-methyl group had effects similar to those observed at the -receptor. These results demonstrate that 7,8-saturated codeine congeners are more efficacious than codeine, which may explain their lack of requirement for 3-O-demethylation in vivo. Nonetheless, because all 7,8-saturated codeine congeners were significantly less potent than their morphine derivatives, further research is needed to understand the relationship between metabolism and in vivo activity of these compounds.The opioid analgesic, codeine (3-methoxymorphine), undergoes several metabolic routes including N-and O-demethylation, as well as glucuronidation (Dayer et al., 1988;Mikus et al., 1991;Caraco et al., 1996;Yu et al., 2002). It is well established that codeine must be O-demethylated to morphine to produce analgesia in both humans (Chen et al., 1991) and rats (Mikus et al., 1991;Cleary et al., 1994). In humans, cytochrome P450 -2D6 (CYP-2D6) catalyzes this reaction, and individuals with dysfunctional allelic variants of this gene are phenotypically described as poor metabolizers (PMs) and are less sensitive to codeine. Individuals expressing multiple copies of active CYP-2D6 are extensive metabolizers (EMs) and are more sensitive to codeine. EMs can exhibit the PM phenotype, however, when codeine is coadministered along with a CYP-2D6 inhibitor such as quinidine. The requirement for O-demethylation is not surprising given that the reported K i values for binding to -opioid receptors is 200 times higher for codeine than for morphine (Chen et al., 1991...