The biological effects of air pollution are still not well known, due to its complex mixture of particulate and gaseous compounds. In vitro cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to in vivo experiments to assess the effects of outdoor air pollution. This study compares two bronchial cell lines, Calu-3 and BEAS-2B, and two alveolar cell lines, hAELVi and A549, regarding their capacity to form a tight epithelial cell barrier for a 2-week culture period and metabolize xenobiotics actively. Culture at the air-liquid interface permits the Calu-3 and hAELVi cells to form and maintain a tight epithelial cell barrier with lower permeability to lucifer yellow, greater trans-epithelial electrical resistance, and the presence of Zonula Occludens 1 (ZO-1) protein at the membrane, than the BEAS-2B and A549 cells. Exposure to benzo(a)pyrene (BaP) induces the up-regulation of CYP1A1 and CYP1B1 genes, proteins, and functional activity at the air-liquid interface in all cell lines. So, these results demonstrate that the Calu-3 and the hAELVi cells are the more relevant models to assess the effects of ambient air pollution at the air-liquid interface, forming a tight epithelial cell barrier and being metabolically active.