Inhibition of inositol 1,4,5-triphosphate (Ins(1,4,5)P3) binding by Ca2+ and Mg2+ remains a controversial issue: the Ca2+ effect may also be explained by Ins(1,4,5)P3 generation due to a Ca(2+)-sensitive phospholipase C, and the Mg2+ effect by Ins(1,4,5)P3 degradation. In this study, we have, therefore, investigated the effects of Ca2+ and Mg2+ on Ins(1,4,5)P3 binding to membranes of the myeloid cell line HL-60 and assayed in parallel phospholipase C activity and Ins(1,4,5)P3 degradation. The assays for Ins(1,4,5)P3 binding, Ins(1,4,5)P3 generation, and Ins(1,4,5)P3 degradation were performed under identical experimental conditions. Ca2+ significantly and reversibly decreased Ins(1,4,5)P3 binding at submicromolar concentrations. Ca2+ stimulated Ins(1,4,5)P3 generation only at concentrations above 1 microM. At 1 microM [Ca2+], Ins(1,4,5)P3 binding was inhibited by 46 +/- 6%, but no Ins(1,4,5)P3 generation was observed. [Mg2+] between 0 and 1 mM slightly stimulated, while higher concentrations inhibited, Ins(1,4,5)P3 binding. Mg2+ did not activate phospholipase C. Neither Ca2+ nor Mg2+ induced relevant Ins(1,4,5)P3 degradation under the conditions of the binding assay. The effects of Ca2+ and Mg2+ on Ins(1,4,5)P3 binding were conserved after solubilization of HL-60 membranes. However, only Mg2+, but not Ca2+, inhibited Ins(1,4,5)P3 binding to the receptor that had been partially purified by heparin affinity chromatography. The Ca2+ sensitivity of Ins(1,4,5)P3 binding to the partially purified receptor could be reconstituted by addition of the flow-through of the heparin column. No Ca(2+)-dependent Ins(1,4,5)P3 generation was observed in the reconstituted system. Thus, we conclude that both Ca2+ and Mg2+ can inhibit Ins(1,4,5)P3 binding independently of phospholipase C activation and Ins(1,4,5)P3 breakdown. The Ca2+ effect depends upon factors that can be separated biochemically from the Ins(1,4,5)P3 receptor. Mg2+ acts either directly on the Ins(1,4,5)P3 receptor or on a closely associated target.