Inositol 1,4,5-trisphosphate receptors (IP ₃ Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca ²⁺ ) signals in response to various external cues. Notably, IP ₃ R channel activity is determined by several obligatory factors, including IP ₃ , Ca ²⁺ , and ATP. The critical basic amino acid residues in the N-terminal IP ₃ -binding core (IBC) region that facilitate IP ₃ binding are well characterized. In contrast, the residues conferring regulation by Ca ²⁺ have yet to be ascertained. Using comparative structural analysis of Ca ²⁺ -binding sites identified in two main families of intracellular Ca ²⁺ -release channels, ryanodine receptors (RyRs) and IP ₃ Rs, we identified putative acidic residues coordinating Ca ²⁺ in the cytosolic calcium sensor region in IP ₃ Rs. We determined the consequences of substituting putative Ca ²⁺ binding, acidic residues in IP ₃ R family members. We show that the agonist-induced Ca ²⁺ release, single-channel open probability (P ₀ ), and Ca ²⁺ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues is neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca ²⁺ -binding pocket shifted the Ca ²⁺ required to activate IP ₃ R to higher concentrations, indicating that these residues likely are a component of the Ca ²⁺ activation site in IP ₃ R. Taken together, our findings indicate that Ca ²⁺ binding to a well-conserved activation site is a common underlying mechanism resulting in increased channel activity shared by IP ₃ Rs and RyRs.