The interaction of inhaled drug molecules with transporter proteins in the airway epithelium is poorly understood, and its clinical significance is largely unknown. This series of experiments aimed to investigate if inhaled drug compounds interact with transporter-mediated trafficking by similar mechanisms in the bronchial and alveolar epithelium. Furthermore the impact of continuous exposure to low concentrations of inhaled therapeutics on drug accumulation in in vitro airway epithelial models was assessed. Both salbutamol and fluticasone significantly increased the accumulation of the P-gp substrate rhodamine-123 (rh-123) in Calu-3 and A549 cells after 17 days in culture. Whilst the increased rh-123 accumulation is likely to be mediated by inhibition of the efflux transporter P-glycoprotein (P-gp) in A549 cells, in Calu-3 cells the data indicated that rh-123 accumulation was driven by an energy-independent and verapamil-insensitive pathway. A549 cells exposed to 100 μM salbutamol and fluticasone for 14 days showed a significant increase (p<0.01) in rh-123 accumulation by 196±3.2 and 158±2.8% respectively. This work suggests that inhaled drugs do impact the transporter-mediated trafficking of compounds in both the alveolar and bronchial epithelium, but are likely to do so through different mechanisms. Furthermore, these studies provide evidence that long term exposure to inhaled therapeutics may impact airway pharmacokinetics, however further studies need to be conducted to establish the specific mechanisms involved and potential transporters implicated in these findings.