Irradiation of a subpopulation of cells is known to cause damage to neighboring cells that are not directly exposed to the radiation, the so-called bystander response. Bystander responses are important when considering the potential damage caused by environmental exposure to radiation or to radiation used in cancer treatment. Although it is known that cells whose nuclei were exposed to radiation can trigger the bystander response, Shao et al. now show that even radiation delivered to the cytoplasm of a single cell triggers bystander responses. To deliver such a targeted irradiation, the authors used a charged-particle microbeam to expose cultured human glioblastoma T98G cells to a controlled number of 3He2+ ions. The bystander response was based on the production of micronuclei (MN) (a measure of chromosomal damage) in the cell population. The abundance of MN increased when a single T98G cell in the population was exposed to a single 3He2+ ion, and the bystander response occurred in T98G cultures and in cocultures with human primary fibroblast AG0 cells. The bystander response was blocked by addition of a nitric oxide (NO) scavenger to the medium. Disruption of glycosphingolipid-enriched membrane microdomains (GEMs) with filipin also blocked the bystander response, which suggests that signals from the membrane are important. When a chemical assay for NO production was used, an increase in the number of cells positive for NO increased after a single T98G cell was exposed to a single 3He2+ ion through the cytoplasm, and this increase in NO was blocked if cells were first treated with filipin. Thus, NO, produced through a process that requires GEMs in response to cytoplasmic irradiation, appears to mediate a signal that causes chromosomal damage in neighboring cells.