Inositol 1,4,5-trisphosphate receptors (InsP₃Rs) are widely-expressed intracellular channels that release Ca²⁺ from internal stores in response to many physiological stimuli. It is becoming apparent that InsP₃Rs form complexes with a multitude of accessory proteins. Many of the proteins that bind to InsP₃Rs appear to modulate channel function and Ca²⁺ release. InsP₃Rs are known to be regulated by the ubiquitous EF-hand Ca²⁺-binding protein calmodulin (CaM). We recently demonstrated that members of the neuronal ‘calcium binding protein’ family (CaBPs), which also bind Ca²⁺ using EF-hand motifs and have approximately 50% homology with CaM, functionally interact with InsP₃ receptors (InsP₃R) and inhibit IICR (1). In the present study, we examined the putative interaction of another EF-hand-containing protein, calmyrin, which is ubiquitously expressed and has 56% homology to CaM. We found that calmyrin interacts with the NH₂-terminus of InsP₃Rs in a Ca²⁺-independent manner (n=3). Video imaging of fura-2 loaded COS7 cells over-expressing YFP-tagged calmyrin revealed that calmyrin inhibited Ca²⁺ release from internal stores induced by purinergic agonist (ATP; 0.5, 1 and 100 µM) (refer to table 1 for percentage of responding cells, peak amplitude of the response and integrated response). A mutated protein in which the consensus site for myristoylation was altered (‘calmyrin-G2A’) also inhibited IICR. Unlike wild-type calmyrin, which was largely bound to cellular membranes, the calmyrin-G2A mutant was diffuse. From these data we concluded that calmyrin function is not dependent on its myristoylation and hence membrane targeting. Recombinant calmyrin also inhibited InsP₃-dependent ⁴⁵Ca²⁺ flux from permeabilised cells (the IC₅₀ of IICR increased from 0.45 ± 0.06 µM to 0.87 ± 0.07 µM in the presence of 10 µM calmyrin (n=3)). The calmyrin-mediated inhibition of Ca²⁺ release was InsP₃R specific since there was no inhibition of caffeine-induced Ca²⁺ release from ryanodine receptors (peak amplitude of response to 0.5 mM caffeine: 59 ± 8.05 nM (n=10 calmyrin-expressing cells) vs. 67.5 ± 4.69 nM (n=42 control cells); p>0.01). To investigate the mechanism by which calmyrin mediates its effect we performed an InsP₃ binding assay using recombinant InsP₃R ligand binding domain. In these studies we demonstrated that calmyrin inhibited InsP₃ binding to the InsP₃R by 22 ± 3% (n=3), in a Ca²⁺-independent manner. Our data indicate that calmyrin interacts with InsP₃Rs and inhibits IICR in a similar manner to CaBPs and CaM.