Nanoparticles (NPs) are included in a variety of consumer products including cosmetics, food, and food packaging. They are also used in medical products for dermal and oral application and for inhalation. The thinness of the air-blood barrier, the large absorption area of the lung, and the relatively low inactivation by enzymes provide fast entry to the systemic blood circulation at high drug concentrations. In addition to intended uptake, exposure to airborne particles from the environment and to NPs released during the manufacturing process may occur. Cytotoxicity is routinely studied for 4-48 h of exposure, but NPs may accumulate in cells and can cause cellular effects after longer times. Both extent and consequences of cellular NP accumulation are currently largely unknown. In vitro studies could help estimating the extent and the consequences of cellular accumulation and classify NPs according to their potential to cause adverse effects upon chronic exposure. Furthermore, such information could help to decrease the amount of labor-and cost-intensive and ethically problematic animal studies. Important parameters for representative chronic cytotoxicity testing in vitro include choice of the appropriate cells and of physiological relevant culture and exposure conditions, verification of uptake, and identification of cell damage. Calu-3 bronchial epithelial cells are a suggested cell line for physiologically relevant testing when cultured on membranes, which allows supplying the cells with nutrients only from the basal side (culture at an air-liquid interface). This culturing also enables the exposure to NPs by aerosol. Cytotoxicity can be identified by changes in cell number, membrane integrity, amount of DNA or protein, and metabolic activity.