The mechanisms of the medium-mediated bystander response induced by gamma-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu-Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of gamma-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2h with CM, 9-11% of cells showed gamma-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for gamma-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.