Traffic-related air pollution is a major public health concern, contributing to respiratory and cardiovascular diseases worldwide. The aim of this study was to investigate the feasibility of using a mobile Air-Liquid Interface (ALI) system to assess the cytotoxicity and inflammatory potential of freshly generated PM2.5 (particle matter with aerodynamic diameter <2.5 mu m) in a road tunnel in Stockholm. We hypothesized that cellular effects would be detectable at lower doses compared to submerged exposures. The mean particle dose in ALI was 1.4 +/- 0.8 mu g/cm(2), whereas a wide range of doses was used for submerged exposures. ALI and submerged results showed that PM2.5 from the road tunnel did not affect the viability of A549 cells, whereas a significant and dose-dependent decrease in viability of dTHP-1 (in submerged exposure) was observed. Furthermore, in A549 in ALI a slight increase in inflammatory response (IL-8, IL-6, and IL-1 beta) was observed. In submerged exposure, the inflammatory response was clearer, particularly in the dTHP-1 cells. In conclusion, this study presents the first successfully conducted in situ ALI exposure in a road tunnel. The results demonstrate that dTHP-1 cells exhibit clear cytotoxic and inflammatory responses, while A549 show only weak effects. These findings suggest that co-cultures of A549 and dTHP-1 may be valuable in future ALI studies.