Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are tetrameric intracellular Ca²⁺-release channels with each subunit containing a binding site for IP₃ in the amino terminus. We provide evidence that four IP₃ molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca²⁺ release suggested that IP₃Rs are maximally occupied by IP₃ before substantial Ca²⁺ release occurs. We showed that ligand binding–deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP₃R subunits, and confirmed the same effect in an IP₃R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP₃Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP₃Rs with four ligand-binding sites exhibited Ca²⁺ release and electrophysiological properties of native IP₃Rs. However, IP₃ failed to activate IP₃Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP₃Rs containing two monomers of IP₃R2_short, an IP₃ binding–deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding-competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP₃-induced Ca²⁺ release only occurs when each IP₃R monomer within the tetramer is occupied by IP₃, thereby ensuring fidelity of Ca²⁺ release.