In this study, we explored the relationship between regulation of surface mu-opioid receptor number, ligand-induced G protein activation (measured by [(35)]S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding) and second messenger signaling (measured by the inhibition of cAMP accumulation). Etorphine and two isomers of cis-beta-hydroxy-3-methylfentanyl (RTI-1a and RTI-1b), which were full agonists for G protein activation and signaling, caused approximately a 50% loss of surface receptors after 1 h of treatment. Fentanyl and morphine were full agonists for inhibiting cAMP accumulation and partial agonists for stimulating [(35)S]GTPgammaS binding and internalization. Although both agonists were approximately 80% as efficacious as etorphine in stimulating [(35)S]GTPgammaS binding, fentanyl induced a 35% loss of surface receptors, whereas morphine only caused a 10% loss. Additionally, both long- and short-term treatment with the opioid antagonist naloxone caused increases in surface receptors. Unexpectedly, the weak partial agonists buprenorphine and one isomer of cis-beta-hydroxy-3-methylfentanyl (RTI-1d) also were found to cause an increase in surface receptors. Treatment with pertussis toxin (PTX) diminished agonist-induced loss of surface receptors. Furthermore, the abilities of morphine and fentanyl to cause internalization were more impaired after PTX treatment than that of etorphine. PTX treatment also significantly enhanced the increase in surface receptor number caused by 18-h treatment with naloxone and buprenorphine. The results of this study suggest that disruption of G protein coupling by PTX treatment affects ligand-regulated mu-receptor trafficking and that partial agonists for signaling can vary greatly in the ability to regulate the number of surface mu-opioid receptors.