The initiation and the early process of bystander response induced by low dose alpha-particle irradiation are very important for understanding the mechanisms underlying the bystander response. Using a 1 cGy alpha-particle to irradiate 50% of the area of a rectangular mylar dish, time-dependent DNA double strand breaks (DSBs) were induced shortly after irradiation in AG1522 cells, located either in the irradiated area or in the non-irradiated bystander area, reaching a maximum 30 min post irradiation. Medium transfer experiments showed that the conditioned medium harvested from the irradiated culture induced excessive DNA DSBs in the medium recipient cells, and the DSB-inducing ability of the medium showed was time-dependent. The medium transfer results indicated that the soluble bystander signalling molecule(s) had been generated very soon (probably less than 2.5 min) after irradiation and exist continuously to 30 min although the production of signalling molecule(s) decreased after 10 min post irradiation. Pre-treatment with dimethyl sulphoxide (DMSO) eliminated the DNA DSB-inducing ability of the conditioned medium, as well as the formation of excessive DNA DSBs in both irradiated and non-irradiated bystander areas, indicating that reactive oxygen/nitrogen species etc. might be involved in these processes.