To assess the toxicity of nanomaterials, most in vitro studies have been performed under submerged conditions, which do not reflect physiological conditions upon inhalation. An air-liquid interface (ALI) exposure may provide more reliable data on dosimetry and prevent interactions with cell culture media components. Therefore, an ALI exposure was combined with a high-throughput RT-qPCR approach to evaluate the toxicological potential of CuO and TiO2 nanoparticles (NP) in A549 cells. While TiO2 NP did not show any cytotoxicity or other effects compromising genomic stability up to 25.8 μg/cm2, CuO NP revealed a dose-dependent cytotoxicity, starting at 4.9 μg/cm2. Furthermore, CuO NP altered distinct gene expression patterns indicative for disturbed metal homeostasis, stress response, and DNA damage induction. Thus, induction of metal homeostasis associated genes (MT1X, MT2A) at 0.4 μg/cm2 and higher suggested uptake and intracellular dissolution of CuO NP, which was verified by a dose-dependent increase in intracellular copper concentration. Starting at 4.9 μg/cm2, oxidative stress markers (HMOX1, HSPA1A) were induced dose-dependently, supported by elevated ROS levels. Furthermore, a dose-dependent induction of genes associated with DNA damage response (DDIT3, GADD45A) was observed, in concordance with an increase in DNA strand breaks. Finally, transcriptional data suggested the induction of apoptosis at high doses, while flow cytometric analysis revealed increased numbers of either late apoptotic or necrotic cells and clearly necrotic cells at the highest concentrations. Thus, an ALI cell culture system was successfully combined with a comprehensive high-throughput RT-qPCR system, allowing the quantification of NP deposition and their impact on genomic stability. For CuO NP, in principle the data confirm observations made under submerged conditions with respect to intracellular copper ion release, as well as oxidative and genotoxic stress response. However, the results derived from ALI exposure allow the assessment of dose-response-relationships as well as the comparison of relative toxic potencies of different NP.