Using micronucleus formation and clonogenic cell survival as endpoints, medium-mediated bystander effects in H460 non-small cell lung cancer cells induced by X-ray irradiation was studied adopting medium transfer method. Differential centrifugation method was used to isolate and purify exosomes from the media of unirradiated and irradiated H460 cells. The morphology of exosomes was observed using transmission electron microscope, the size distribution of exosomes was measured using Zetasizer Nano ZS 90 size detector, and the expression of a marker of exosomes, hsp90β, was detected by Western blot. Fluorescence probes were applied to investigate the internalization of exosomes into recipient cells. The effects of exosomes on the proliferation of recipient cells were assessed using crystal violet assay. The data showed that X-rays could induce medium-mediated bystander effects in H460 cells manifesting as an increase in micronucleus formation and a decrease in clonogenic cell survival of bystander cells. Both unirradiated and irradiated H460 cells secreted exosomes, but with different size distribution. When incubated with recipient cells, exosomes could be internalized quickly into recipient cells probably through membrane fusion, promoting proliferation in recipient cells. The exosomes secreted by irradiated cells had no effects on the clonogenic cell survival rate of recipient cells, while they could induce an increase in the frequency of micronucleus formation in recipient cells, which could be abolished by the pretreatment of exosomes with RNase. These results suggest that exosome secreted by irradiated cells might be one of the mechanisms underlying RIBEs, and RNAs in exosomes might play an important role.Cited