The β2-AR (β2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of β2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2-AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2-AR (designated β2-AR-RE, β2-AR-GE, β2-AR-RQ and β2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg16 (β2-AR-RE and β2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (β2-AR-GE and β2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β2-AR-RE relative to β2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of β2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of β2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.