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Epidemiological studies have shown an association between airborne particles and a wide range of adverse health effects. The mechanisms behind these effects include oxidative stress and inflammation. Even though traffic gives rise to high levels of particles in the urban air, people are exposed to even higher levels in the subway. However, there is a lack of knowledge regarding how particles from different urban subenvironments differ in toxicity. The main aim of the present study was to compare the ability of particles from a subway station and a nearby very busy urban street, respectively, to damage DNA and to induce oxidative stress. Cultured human lung cells (A549) were exposed to particles, DNA damage was analyzed using single cell gel electrophoresis (the comet assay), and the ability to induce oxidative stress was measured as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in lung cell DNA. We found that the subway particles were approximately eight times more genotoxic and four times more likely to cause oxidative stress in the lung cells. When the particles, water extracts from the particles, or particles treated with the metal chelator deferoxamine mesylate were incubated with 2'-deoxyguanosine (dG) and 8-oxodG was analyzed, we found that the oxidative capacity of the subway particles was due to redox active solid metals. Furthermore, analysis of the atomic composition showed that the subway particles to a dominating degree (atomic %) consisted of iron, mainly in the form of magnetite (Fe3O4). By using electron microscopy, the interaction between the particles and the lung cells was shown. The in vitro reactivity of the subway particles in combination with the high particle levels in subway systems give cause of concern due to the high number of people that are exposed to subway particles on a daily basis. To what extent the subway particles cause health effects in humans needs to be further evaluated.