Diesel emissions are the main source of air pollution in urban areas, and diesel exposure is linked with substantial adverse health effects. In vitro diesel exposure models are considered a suitable tool for understanding these effects. Here we aimed to use a controlled in vitro exposure system to whole diesel exhaust to study the effect of whole diesel exhaust concentration and exposure duration on mucociliary differentiated human primary bronchial epithelial cells (PBEC). PBEC cultured at the air-liquid interface (ALI) were exposed for 60 to 375 minutes to three different dilutions of Diesel Exhaust (DE). The DE mixture was generated by an engine at 47 % load, and characterized for particulate matter (PM) size and distribution, chemical and gas composition. Cytotoxicity and epithelial barrier function was assessed, as well as mRNA expression and protein release analysis. DE caused a significant dose-dependent increase in expression of oxidative stress markers (HMOX1, and NQO1; n=4) at 6 h after 150 min exposure. Furthermore, DE significantly increased the expression of the markers of the integrated stress response (ISR) CHOP and GADD34 and of the pro-inflammatory chemokine CXCL8, as well as release of CXCL8 protein. Cytotoxic effects or effects on epithelial barrier function were observed only after prolonged exposures to the highest DE dose. These results demonstrate the suitability of our model, and that exposure dose and duration, and time of analysis post-exposure are main determinants for the effects of diesel exhaust on differentiated primary human airway epithelial cells.