Ca2+ release from intracellular stores occurs via two families of intracellular channels, each with their own specific agonist: Ins(1, 4,5)P3 for the Ins(1,4,5)P3 receptor and cyclic ADP-ribose (cADPR) for the ryanodine receptor. We now report that cADPR inhibited Ins(1, 4,5)P3-induced Ca2+ release in permeabilized A7r5 cells with an IC50 of 20 microM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 microM. This inhibition was accompanied by an increase in specific [3H]Ins(1,4,5)P3 binding. 8-Amino-cADPR, but not 8-bromo-cADPR, antagonized this effect of cADPR. The inhibition was prevented by a whole series of inositol phosphates (10 microM) that did not affect Ins(1,4,5)P3-induced Ca2+ release, and by micromolar concentrations of PPi and various nucleotide di- or triphosphates. We propose that cADPR must interact with a novel regulatory site on the Ins(1,4,5)P3 receptor or on an associated protein. This site is neither the Ins(1,4,5)P3-binding domain, which prefers Ins(1,4,5)P3 and only binds nucleotides and PPi in the millimolar range, nor the stimulatory adenine nucleotide binding site.