Calcium is required as a cofactor by primer recognition proteins involved in DNA synthesis and by protein kinase C (PKC), which is activated by ionizing radiation. Because these processes may be involved in radiation- mediated regulation of the progression of cells through the phases of the cell cycle, we studied the effects of the intracellular Ca2+ chelator, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (AM- BAPTA), on PKC activation, expression of c-jun and Gadd45 and distribution of cells in the phases of the cell cycle after irradiation. AM-BAPTA prevented ionizing-radiation-induced activation of PKC and expression of c-jun in cells of human tumor cell lines. Conversely, calcium chelation had no effect on X- ray-induced expression of the Gadd45 gene. To determine whether changes in the intracellular Ca2+ concentration ([Ca2+](i)) occurred during irradiation, we measured [Ca2+](i) in single cells using fura-2-based microfluorimetry. There was no increase in [Ca2+](i) during or after irradiation of cells of the human tumor cell lines RIT-3, SQ-20B or HL-60 or normal human fibroblast strain IMR-90. The percentage of human tumor cells crossing the G1/S-phase border was reduced by pretreatment with AM-BAPTA. These data indicate that calcium is required for ionizing radiation-induced cell cycle regulation and PKC activation, but that increases in [Ca2+](i) do not occur in cells of the cell lines irradiated in this study.