AbstractIonizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca²⁺-activated-K⁺-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H₂O₂ (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H₂O₂ in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H₂O₂ in the nucleus and in the cytosol. This rise, which is determined by the rate of H₂O₂ production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca²⁺ and eventually an activation of hIK channels.