Cell cycle arrest and induction of the DNA-repair machinery promotes radioresistance of tumor cells subjected to ionizing radiation (IR). To define the role of Ca²⁺ signaling in this process, K562 leukaemia cells were irradiated (0 and 10 Gy) and cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane cation conductance, CaMKII activity, and cell cycle were monitored by patch clamp whole-cell recording, Fura-2 Ca²⁺ imaging, immunoblotting and flow cytometry (Nicoletti staining), respectively. As a result, IR stimulated a larger increase in [Ca²⁺]_i after Ca²⁺ re-addition to the medium in Ca²⁺-depleted K562 cells. Depletion of the intracellular Ca²⁺ stores by thapsigargin (1 mM) did not further increase the IR-stimulated Ca²⁺ leak across the plasma membrane suggesting a Ca²⁺ store-independent activation of Ca²⁺-permeabel channels by IR. Accordingly, patch-clamp recording demonstrated IR-stimulated activation of non-selective cation channels which generated significantly higher Ca²⁺-carried inward currents as compared to the non-irradiated control cells. Moreover, IR stimulated a modification of the activated CaMKII (alpha isoform) and a G2/M cell cycle arrest. To test for a functional significance of Ca²⁺ entry, K562 cells were irradiated and further incubated in the presence of EGTA (3 mM) and ionomycin (1 mM) and the cell cycle was analyzed by flow cytometry. Ca²⁺ depletion impaired IR-stimulated cell cycle arrest resulting in pronounced IR-induced cell death while having no effect on non-irradiated cells. In conclusion, IR-stimulated cation channel activation and Ca²⁺ entry participate in cell cycle arrest of irradiated K562 cells.