Agonist-activated beta2-adrenoceptors rapidly internalize and then recycle to the cell surface, however chronic agonist eventually causes receptor downregulation. To characterize beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation, human embryonic kidney cells expressing epitope-tagged receptors were examined by radioligand binding with (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on hydrochloride ([3H]CGP12177) and immunofluorescence microscopy. The first-order receptor recycling rate constant declined after 18 h of agonist compared with 15 min (0.05 min(-1) vs. 0.12 min(-1)), thus increasing the intracellular transit time (20.0 min vs. 8.3 min). There was also a reduction in the rate of receptor endocytosis and a decline in the total number of receptors. Although the intracellular receptor fraction did not increase between 15 min and 18 h of agonist, some receptors moved irreversibly into a protease-containing compartment while retaining radioligand binding activity. Our results indicate that beta2-adrenoceptor downregulation is associated principally with an increased intracellular transit time during recycling. This could promote the diversion of receptors into protease-containing compartments, where there is an irreversible commitment to downregulation prior to loss of radioligand binding activity.