Nanoparticles (ultrafine particles) are prevalent in various environments and raise concerns due to their potential health effects. In this study, we aimed to enhance the understanding of the toxicity associated with nanoparticles generated within subway systems. Specifically, we investigated nanoparticles produced using spark discharge from electrodes made of the same material as the third rail (which provides electric power), rail, and wheel components in the Stockholm subway system. Characterization revealed that the generated nanoparticles typically had a primary size of 6–10 nm and exhibited high agglomeration. They consisted mainly of iron, along with varying amounts of manganese and silicon. Despite having low oxidative potential, they showed some cytotoxicity and clearly induced DNA strand breaks in both dTHP-1 cells (monocyte-derived macrophages) and A549 cells (lung epithelial cells). In addition, gene expression analysis showed an upregulation of the cytokine IL-8 in dTHP-1 cells. No increased release of IL-1β, IL-8, IL-6, and TNF-a was noted. Consistent differences in toxicity between the nanoparticles from different materials were not observed. In conclusion, the results show that subway-related nanoparticles can cause DNA damage in cultured lung cells, but the inflammatory potential in terms of cytokine release was limited.