The human bronchiolar epithelial cell line, NCI-H441 has recently been introduced as an in vitro model for studying drug disposition at the distal lung air-blood barrier. Here, we determined whether levels of expression and function of major ATP-binding cassette transporters, P-glycoprotein (P-gp) and multidrug resistance-related protein-1 (MRP1) in HCI-H441 cells were comparable to those in freshly isolated human distal lung epithelial cells in primary culture. P-gp and MRP1 expression was quantified by immunoblot, whilst confocal laser scanning microscopy (CLSM) was employed to determine transporter localisation. Efflux experiments with rhodamin 123 (Rh123, P-gp substrate) and 5,6-carboxyfluorescein-diacetate (CF, MRP1 substrate) were carried out assessing transporter function in vitro. Furthermore, the inhibitory effects of LY-335979 (specific P-gp inhibitor) and MK-571 (specific MRP1 inhibitor) on Rh123 and CF efflux were investigated. Immunoblot provided evidence that NCI-H441 cell monolayers expressed both efflux pumps on protein level. CLSM data confirmed these observations and moreover, revealed localisation of the transporters along apical membranes and in perimembranous vesicles. Rh123 and CF were both released from NCI-H441 monolayers in a time-dependent manner. This efflux could be inhibited by the relevant pharmacological agents. P-gp and MRP1 expression and activity in NCI-H441 cell monolayers were found to be consistent with data previously reported from human peripheral lung epithelial cells in primary cultures. The use of NCI-H441 cells to investigate efflux pump effects at the air-blood barrier can therefore be recommended.