Abstract Carbon nanotubes (CNTs) represent one of the most promising engineered nanomaterials, with possible applications in advanced engineering and biomedical technologies. During their production, human exposure to CNTs may occur via inhalation. Therefore, the aim of this study was to mimic inhalation of multi-walled CNTs (MWCNTs) in vitro as realistically as possible, by producing MWCNTs aerosols via an Air-Liquid Interface Cell Exposure System (ALICE), combined with a 3D epithelial airway barrier model cultivated at the air-liquid interface (ALI). To address the consequences of an extended exposure period, repeated exposures of MWCNTs (total deposition 1.15 μg/cm(2)) were applied to the co-culture system, either over one day (one day repeated exposure) or three days (three day repeated exposure scenario). Although in both repeated exposure scenarios MWCNTs were found to interact with the different cell types, they did not induce any cytotoxicity or alterations in cell morphology, nor did they elucidate any significant increase in pro-inflammatory markers compared to control cultures. Similar results were also observed following single MWCNTs exposures at deposited concentrations of 0.14, 0.20 and 0.39 µg/cm(2). Cells exposed repeatedly to MWCNTs for three days, however did show a decrease in reduced glutathione levels, although not significant (p > 0.05). In conclusion, we have presented a realistic in vitro alternative to mimic occupational exposure of MWCNTs and by applying this approach it was shown that repeated MWCNT exposures to lung cell cultures at the ALI elicit a limited biological impact over a three day period.