Patients who undergo external beam radiotherapy are at risk of developing second tumours due to scattered radiation outside the path of the primary beam. The aim of this study was to experimentally determine the in vitro radiobiological effects of scattered radiation in cells located outside the primary photon beam and to compare this to the effects that occur in cells inside the primary beam. The comparison was performed by assessing cell viability, DNA damage, and apoptosis.Cells from the human breast cancer line MDA-MB-231 were inserted in a water phantom and irradiated at varying doses (1.5, 2.0, 2.5, and 3.0Gy). The cells were placed at two geometrical points: in the central beam axis and at 10cm out-of-field. The dose was constant in both geometrical points. Survival fraction, number of DNA double strand-breaks, and cleaved poly-(ADP-ribose) polymerase (PARP) levels were determined by clonogenic assay and flow cytometry.A slight, non-significant decrease of 3 to 5% in cell survival fraction was observed in cells irradiated outside the primary field. The number of PARP-positive cells and DNA double strand-breaks both increased after out-of-field irradiation.Scattered irradiation appears to induce an in vitro biological response on out-of-field cells that is stronger than the effect of primary radiation on in-field cells, independent of the bystander effect. These findings suggest that the biological response of healthy tissues outside the primary beam might be higher than previously believed.