As air pollution is a major problem in Tehran, this study aimed to investigate the physicochemical characterization of thewater-soluble and organic contents of ambient PM2.5 in Tehran and determine its in vitro toxicological impact on human lung epithelial cells (A549). A total of 11 sampling stations were selected, including three categories: traffic, urban, and suburban. All sampling was carried out in the spring and summer of 2015. Ion chromatography (IC), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and GC‐MS were used to analyze ionic compounds, heavy metals, and polycyclic aromatic hydrocarbons (PAHs), respectively, and an ELISA reader was used for cytotoxicity analysis. The most prevalent ionic species found for all three categories was SO4 2−. PAH concentrations were 43.45 ± 32.71, 50.51 ± 37.27, and 29.13 ± 33.29 ng/m3 for traffic, urban, and suburban stations, respectively. For all sampling stations, Al and Fe had the highest values among the investigated heavy metals. Cell viability measurements, carried out using theMTT assay, showed that all three categories of samples cause cytotoxicity, although the urban station samples showed higher cytotoxicity than those from the other stations (p ˂ 0.05). Based on the results of the present study, organic compounds and insoluble particles could be the main causes of cytotoxicity.