Abstract Especially through the release from anthropogenic sources, there is growing concern about human exposure to ultrafine particulate matter (UFP). Inhalation of UFP is associated with respiratory diseases such as asthma, pulmonary fibrosis and lung cancer, but their mechanisms of action are still vastly unknown. In this study we aimed to address the question whether the physical characteristics of UFP contribute most to their toxicity or whether the particles primarily act as carriers of toxic chemicals. For this scope, two distinct UFP aerosols with low or high content of semi-volatile organic compounds (SVOC) but comparable sizes were produced by a combustion aerosol standard soot generator and investigated regarding their acute (geno)-toxic potential in a cell model consisting of alveolar epithelial cells, macrophages, and fibroblasts at the air-liquid interface. Low SVOC UFP induced an increased cytotoxicity accompanied by a reduced cell number of epithelial cells and a decreased metabolic activity of fibroblasts. Furthermore, an increased intracellular release of reactive oxygen species (ROS) was observed after low SVOC UFP treatment, which resulted in a depletion of intracellular glutathione. The increased ROS release could be causative for the observed secondary genotoxicity in fibroblasts exclusively detected after exposure to low organic content UFP. High SVOC UFP did not impact cell viability, but an increased DNA damage could be detected in epithelial cells. All in all, UFP with low organic content seem to induce a stronger acute cytotoxic and oxidative stress response in epithelial cells, which might cause secondary effects in fibroblasts.