The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 μg ZnO/cm2 (or 0.09 and 0.33 cm2 ZnO/cm2), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue.